This document discusses the history and techniques of immunohistochemistry. It covers:
- The early development of immunohistochemistry from 1941 to the present.
- Common fixatives used such as formalin, Bouin's solution, B5, Zenker's solution, and modifications like PLDP.
- The benefits and drawbacks of different fixatives for preserving tissue morphology and antigenicity.
- Methods for improving antigen retrieval after fixation, like proteolytic enzymes and heat.
- The importance of fixation for maintaining tissue structure while not blocking epitopes.
principle of Immunohistochemistry and its use in diagnosticsEkta Jajodia
Immunohistochemistry (IHC) localizes antigens in tissues based on antigen-antibody recognition. The principle is visualizing target compounds in tissues with high signal-to-noise ratio. IHC was developed in the 1960s using enzyme labels instead of fluorescent labels to visualize targets under a light microscope. Key steps in IHC include antigen retrieval to unmask antigens, blocking endogenous enzymes, primary antibody incubation, secondary antibody or polymer incubation, and signal development with chromogens. IHC is commonly performed on formalin-fixed paraffin-embedded sections and can identify cell types and localization of proteins to characterize tissues.
This is a presentation I prepared to demonstrate my mastery of the basics of Immunohistochemistry during my first two months of employment as a Biologist at the Cell Marque Corporation. Please note, there are a few slides that appear to be dysfunctional and overlapping; this is due to the fact that these particular slides included complex animations that I designed to illustrate various scientific concepts related to the practice of Immunohistochemistry. If you wish to view this presentation in its entirety (animations included), feel free to contact me via LinkedIn and I will gladly provide you with a fully-functional version.
priciples and applications Immunohistochemistry Markos Tadele
This document provides an overview of immunohistochemistry (IHC), including the principle, general protocol, and key steps. IHC combines histology and immunology to identify specific tissue components using antigen-antibody reactions tagged with visible labels. The protocol involves fixing, embedding, sectioning tissues, performing antigen retrieval, incubating with primary/secondary antibodies, and visualizing the antigen-antibody complex through detection systems like peroxidase or fluorescence. Proper controls and interpretation by a pathologist are needed for accurate results.
Histology is the microscopic study of tissues. Key steps in processing tissues for histological examination include fixation, dehydration, clearing, embedding in paraffin wax, sectioning, and staining. Tissues are first fixed in chemicals like formaldehyde to preserve their structure. They are then dehydrated using graded alcohols to remove water. Next, tissues are cleared using solvents like xylene to make them permeable to paraffin prior to embedding. The embedded tissues can then be thinly sectioned and stained for microscopic examination. Proper tissue processing is important for high quality histological analysis.
Immunohistochemistry (IHC) utilizes labeled antibodies to localize specific antigens in tissue sections through antigen-antibody interactions visualized by markers like fluorescent dyes or enzymes. IHC allows visualization of the distribution and localization of cellular components within tissues. The process involves raising antibodies to target antigens, labeling the antibodies, and applying them to tissue sections using techniques like direct, indirect, or peroxidase anti-peroxidase (PAP) methods. IHC is a sensitive and specific technique that is useful for cancer diagnosis and research applications.
Immunohistochemistry utilizes labeled antibodies to localize specific antigens within cells and tissues. It combines histological, immunological, and biochemical techniques to visualize the distribution of cellular components through antigen-antibody reactions tagged with visible labels. Tissues are prepared through fixation and sectioning, then exposed to labeled antibodies targeting specific antigens. This allows visualization of targeted proteins and structures under a microscope. IHC is a sensitive technique useful for cancer diagnosis, differential diagnosis, and research applications.
Flow cytometry is a technique that allows for the analysis of physical and fluorescent characteristics of single cells as they flow in a liquid stream past a laser. It involves using antibodies tagged with fluorescent dyes to detect cell antigens. This allows for the identification of cell types, lineages, and maturation stages. Flow cytometry provides sensitive, quantitative, and multiparameter analysis of cells and is widely used for immunophenotyping in hematological malignancies and other disorders.
principle of Immunohistochemistry and its use in diagnosticsEkta Jajodia
Immunohistochemistry (IHC) localizes antigens in tissues based on antigen-antibody recognition. The principle is visualizing target compounds in tissues with high signal-to-noise ratio. IHC was developed in the 1960s using enzyme labels instead of fluorescent labels to visualize targets under a light microscope. Key steps in IHC include antigen retrieval to unmask antigens, blocking endogenous enzymes, primary antibody incubation, secondary antibody or polymer incubation, and signal development with chromogens. IHC is commonly performed on formalin-fixed paraffin-embedded sections and can identify cell types and localization of proteins to characterize tissues.
This is a presentation I prepared to demonstrate my mastery of the basics of Immunohistochemistry during my first two months of employment as a Biologist at the Cell Marque Corporation. Please note, there are a few slides that appear to be dysfunctional and overlapping; this is due to the fact that these particular slides included complex animations that I designed to illustrate various scientific concepts related to the practice of Immunohistochemistry. If you wish to view this presentation in its entirety (animations included), feel free to contact me via LinkedIn and I will gladly provide you with a fully-functional version.
priciples and applications Immunohistochemistry Markos Tadele
This document provides an overview of immunohistochemistry (IHC), including the principle, general protocol, and key steps. IHC combines histology and immunology to identify specific tissue components using antigen-antibody reactions tagged with visible labels. The protocol involves fixing, embedding, sectioning tissues, performing antigen retrieval, incubating with primary/secondary antibodies, and visualizing the antigen-antibody complex through detection systems like peroxidase or fluorescence. Proper controls and interpretation by a pathologist are needed for accurate results.
Histology is the microscopic study of tissues. Key steps in processing tissues for histological examination include fixation, dehydration, clearing, embedding in paraffin wax, sectioning, and staining. Tissues are first fixed in chemicals like formaldehyde to preserve their structure. They are then dehydrated using graded alcohols to remove water. Next, tissues are cleared using solvents like xylene to make them permeable to paraffin prior to embedding. The embedded tissues can then be thinly sectioned and stained for microscopic examination. Proper tissue processing is important for high quality histological analysis.
Immunohistochemistry (IHC) utilizes labeled antibodies to localize specific antigens in tissue sections through antigen-antibody interactions visualized by markers like fluorescent dyes or enzymes. IHC allows visualization of the distribution and localization of cellular components within tissues. The process involves raising antibodies to target antigens, labeling the antibodies, and applying them to tissue sections using techniques like direct, indirect, or peroxidase anti-peroxidase (PAP) methods. IHC is a sensitive and specific technique that is useful for cancer diagnosis and research applications.
Immunohistochemistry utilizes labeled antibodies to localize specific antigens within cells and tissues. It combines histological, immunological, and biochemical techniques to visualize the distribution of cellular components through antigen-antibody reactions tagged with visible labels. Tissues are prepared through fixation and sectioning, then exposed to labeled antibodies targeting specific antigens. This allows visualization of targeted proteins and structures under a microscope. IHC is a sensitive technique useful for cancer diagnosis, differential diagnosis, and research applications.
Flow cytometry is a technique that allows for the analysis of physical and fluorescent characteristics of single cells as they flow in a liquid stream past a laser. It involves using antibodies tagged with fluorescent dyes to detect cell antigens. This allows for the identification of cell types, lineages, and maturation stages. Flow cytometry provides sensitive, quantitative, and multiparameter analysis of cells and is widely used for immunophenotyping in hematological malignancies and other disorders.
This document provides information on immunohistochemistry (IHC), including:
1. IHC is used to detect antigens in tissues through antigen-antibody recognition at the light microscopic level. It applies immunologic principles and techniques to study cells and tissues.
2. The basic principle of IHC is a sharp visualization of target components in cells and tissues based on a satisfactory signal-to-noise ratio.
3. The main steps of IHC are tissue processing, antigen retrieval, primary/secondary antibody incubation, detection, counterstaining, and mounting. Proper controls and interpretation of results are also discussed.
Important auxiliary method for pathologists in routine diagnostic work as well as in basic and clinical research including exploration of biomarkers, as IHC allows confirmation of target molecule expressions in the context of microenvironment.
pathogenesis, diagnosis, and treatment of diseases are discovered.It is performed without destruction of histologic architecture. It can be used as an effective adjuvant to H & E diagnosis in a majority of tumor cases through the establishment of definitive diagnosis and also gives an insight into tumor histopathogenesis and prognosis.
This document discusses antigen retrieval in immunohistochemistry. It begins with a brief history and defines key terms like antigen, antibody, affinity, and sensitivity. It describes how antigens can become masked during tissue fixation and various techniques to retrieve masked antigens, including heat-induced epitope retrieval (HIER), protease-induced epitope retrieval (PIER), and combinations of the two. Specific methods of HIER using microwave ovens, pressure cookers, steamers and other devices are outlined. The document also discusses newer antigen retrieval techniques like those for frozen sections. In summary, antigen retrieval techniques aim to expose masked antigens by reversing the effects of tissue fixation through the use of heat and enzymes.
The document summarizes a seminar presentation on histopathology staining techniques. It discusses the routine H&E stain and provides details on the principle, reagents, and procedure. It also describes special stains used to identify substances like carbohydrates, amyloid, nucleic acids, and lipids. Specific stains covered include periodic acid Schiff, Congo red, Feulgen, methyl green pyronin, and Oil red O. The document aims to inform attendees about common and special staining methods in histopathology.
Immunohistochemistry (IHC) is a technique that combines immunology and histology to detect antigens in tissues using antibodies, with the main steps including preparing tissue sections, using primary and secondary antibodies to target antigens, developing signals using reporters and counterstains, and observing under a microscope. IHC is widely used for disease diagnosis by identifying molecular markers characteristic of cellular events involved in diseases like cancer and neurological disorders. Troubleshooting for IHC focuses on reducing background staining and improving antigen detection through optimizing antibody concentrations and incubation times.
This document discusses antigen retrieval techniques, which are methods used to expose antigens that have become masked during tissue fixation. It describes the principles and procedures of protease-induced epitope retrieval (PIER) using enzymes like trypsin or pepsin, heat-induced epitope retrieval (HIER) using a microwave or pressure cooker, and combinations of heat and enzymes. The staining procedure for LSAB-peroxidase immunohistochemistry is also outlined.
This document discusses immunofluorescence, a technique used to detect antibodies in serum or body fluids. It involves using a primary antibody that binds to the target antigen, then a fluorescent secondary antibody that binds to the primary. This allows visualization under a microscope. Two types are described: direct uses a fluorescent primary antibody, indirect uses a non-fluorescent primary and fluorescent secondary for signal amplification. Applications include detecting autoantibodies associated with various diseases by looking for fluorescence patterns on tissue sections like Hep-2 cells or mouse organs. Indirect immunofluorescence is considered the standard technique as it has high sensitivity and specificity.
Immunohistochemistry (IHC) is a laboratory technique that uses antibodies to detect antigens in tissue samples. The document provides an overview of IHC, including its history, principles, steps, methods, applications and troubleshooting. Key developments include the indirect method in 1942 and enzyme conjugation in 1966. The main steps are tissue collection, fixation, sectioning, antigen retrieval, staining, detection and counterstaining. IHC is useful for cancer prognosis, infectious disease diagnosis and research applications by determining the presence or absence of cell markers.
Immunohistochemistry is a technique used to identify antigens in tissue samples using antigen-antibody interactions. It has made a large impact in disease diagnosis since its development in the 1940s-1970s. The technique involves using labeled antibodies that specifically bind to antigens in tissue sections. This binding is then visualized using markers like enzymes or fluorescent dyes. Several methods have been developed to increase the signal and reduce background noise, including indirect labeling techniques and polymer-based methods. Proper tissue processing and antibody selection are important for obtaining high quality results with immunohistochemistry.
Immunohistochemistry (IHC) is a highly sensitive method that allows the localization of antigen within a cell or a tissue with high resolution. The method is based on the use of a primary antibody that specifically binds to its complementary antigen. The bound antibody may then be visualized by a variety of methods such as colorimetric end points.
THEORIES OF STAINING Biological Staining
Structural Components (Nature) Of Stains
Staining Mechanism
Metachromasia
Types Of Staining
Staining of Paraffin Section
A stain is any colouring organic compound that combined with another substance imparts a colour to that substance.
The term ‘dye’ is used to refer to a colouring agent that is used for general purposes, whereas the term ‘stain’ is used to refer to that dye which is used for biological purposes.
The stains used for bacteria are aniline dyes they are derived from aniline (C6H5NH2).
The most commonly used aniline dyes are crystal violet, methylene blue, basic fuchsin, safranin, eosin, etc.FACTORS INFLUENCING METACHROMASIA
Immunohistochemistry is a technique that uses antibodies to identify antigens in cells of a tissue sample. It relies on the principle of antibodies binding specifically to antigens in cells. The primary antibody binds to the antigen of interest, while the secondary antibody is conjugated to an enzyme or fluorescent label for visualization. A chromogen is used to produce a colored precipitate that can be seen under the microscope. Immunohistochemistry has many applications in pathology for tumor diagnosis and classification by identifying cellular markers of differentiation. It allows identification of cell lineages and tumor types through characteristic protein expression patterns revealed by specific antibody staining.
This document provides an overview of cancer immunology, including how tumors are able to evade and enhance immune responses. It discusses the types of tumors of the immune system, such as lymphomas and leukemias, and how tumors enhance their growth by modulating antigens, expressing low levels of MHC molecules, and lacking costimulatory signals. It also describes tumor antigens, how the immune system responds to tumors through cells like NK cells and macrophages, and immunotherapy approaches to boost anti-tumor immunity.
Enzymes are proteins that catalyze chemical reactions without being changed chemically. They act as catalysts by lowering the activation energy of reactions and increasing their rate. Enzymes are usually named by adding "ase" to the substrate they act on and can be classified into six groups including oxidoreductases, transferases and hydrolases. Enzyme histochemistry is used in muscle disease diagnosis by demonstrating enzyme activity and localization in tissue sections.
The document discusses the history, utility, and methods of preparing cell blocks from fine needle aspiration cytology samples. Cell blocks allow examination of histological structure and use of ancillary tests. Key methods include the fixed sedimentation method using a 1:1 ratio of 100% alcohol and 40% formalin, the plasma thrombin method using equal parts plasma and thrombin, and the bacterial agar method using 3% agar. Cell blocks provide increased diagnostic sensitivity and specificity compared to cytology alone through examination of tissue architecture and ability to perform special stains and molecular testing.
This document provides an overview of immunohistochemistry (IHC), including its history, basic methods, considerations, and applications. IHC combines histology, immunology, and biochemical techniques to visualize the distribution and localization of specific cellular components within tissues through antigen-antibody reactions tagged with visible labels. Key steps discussed include fixation, sectioning, antigen retrieval, blocking, controls, direct and indirect antibody binding methods, detection methods including enzymatic and fluorescence techniques, and important considerations for optimizing IHC experiments.
Immunofluorescence is a technique that uses the binding of antibodies to antigens to detect the presence of substances. It relies on tagging antibodies with fluorescent dyes called fluorophores. When exposed to ultraviolet light, the fluorophore-tagged antibody complexes emit visible light that can be seen using a fluorescent microscope. There are several types of immunofluorescence assays including direct, indirect, and quantitative assays that are used to detect microorganisms, antibodies, and other biomolecules through the antigen-antibody reaction and fluorescent signal produced.
FIXATIVES in Pathology for Postgraduate and DMLTjenishJebadurai1
This document discusses various fixatives used in histology and cytology techniques. It begins by defining fixation as the process of preserving cells and tissue using physical or chemical methods. Commonly used fixatives include formaldehyde, glutaraldehyde, osmium tetroxide, mercury salts, picric acid, and alcohols. An ideal fixative would be non-toxic, low-cost, and effectively preserve tissue morphology, antigens, and nucleic acids while allowing for long-term storage. Factors like temperature, pH, concentration, and duration impact fixation quality. Proper fixation is important for minimizing artifacts and ensuring high quality staining.
The document discusses fixation in histology and cytology. It describes the aims of fixation as preserving tissue structure and preventing autolysis and bacterial growth. Fixation causes some changes like tissue shrinkage and hardening. The types of fixation include immersion, coating, vapor and perfusion. Formalin and alcohol are common fixatives. Factors like pH, temperature, duration and osmolarity influence fixation quality. The choice of fixative depends on the tissue and technique used, for example glutaraldehyde for electron microscopy. Useful fixative formulas are also provided.
This document provides information on immunohistochemistry (IHC), including:
1. IHC is used to detect antigens in tissues through antigen-antibody recognition at the light microscopic level. It applies immunologic principles and techniques to study cells and tissues.
2. The basic principle of IHC is a sharp visualization of target components in cells and tissues based on a satisfactory signal-to-noise ratio.
3. The main steps of IHC are tissue processing, antigen retrieval, primary/secondary antibody incubation, detection, counterstaining, and mounting. Proper controls and interpretation of results are also discussed.
Important auxiliary method for pathologists in routine diagnostic work as well as in basic and clinical research including exploration of biomarkers, as IHC allows confirmation of target molecule expressions in the context of microenvironment.
pathogenesis, diagnosis, and treatment of diseases are discovered.It is performed without destruction of histologic architecture. It can be used as an effective adjuvant to H & E diagnosis in a majority of tumor cases through the establishment of definitive diagnosis and also gives an insight into tumor histopathogenesis and prognosis.
This document discusses antigen retrieval in immunohistochemistry. It begins with a brief history and defines key terms like antigen, antibody, affinity, and sensitivity. It describes how antigens can become masked during tissue fixation and various techniques to retrieve masked antigens, including heat-induced epitope retrieval (HIER), protease-induced epitope retrieval (PIER), and combinations of the two. Specific methods of HIER using microwave ovens, pressure cookers, steamers and other devices are outlined. The document also discusses newer antigen retrieval techniques like those for frozen sections. In summary, antigen retrieval techniques aim to expose masked antigens by reversing the effects of tissue fixation through the use of heat and enzymes.
The document summarizes a seminar presentation on histopathology staining techniques. It discusses the routine H&E stain and provides details on the principle, reagents, and procedure. It also describes special stains used to identify substances like carbohydrates, amyloid, nucleic acids, and lipids. Specific stains covered include periodic acid Schiff, Congo red, Feulgen, methyl green pyronin, and Oil red O. The document aims to inform attendees about common and special staining methods in histopathology.
Immunohistochemistry (IHC) is a technique that combines immunology and histology to detect antigens in tissues using antibodies, with the main steps including preparing tissue sections, using primary and secondary antibodies to target antigens, developing signals using reporters and counterstains, and observing under a microscope. IHC is widely used for disease diagnosis by identifying molecular markers characteristic of cellular events involved in diseases like cancer and neurological disorders. Troubleshooting for IHC focuses on reducing background staining and improving antigen detection through optimizing antibody concentrations and incubation times.
This document discusses antigen retrieval techniques, which are methods used to expose antigens that have become masked during tissue fixation. It describes the principles and procedures of protease-induced epitope retrieval (PIER) using enzymes like trypsin or pepsin, heat-induced epitope retrieval (HIER) using a microwave or pressure cooker, and combinations of heat and enzymes. The staining procedure for LSAB-peroxidase immunohistochemistry is also outlined.
This document discusses immunofluorescence, a technique used to detect antibodies in serum or body fluids. It involves using a primary antibody that binds to the target antigen, then a fluorescent secondary antibody that binds to the primary. This allows visualization under a microscope. Two types are described: direct uses a fluorescent primary antibody, indirect uses a non-fluorescent primary and fluorescent secondary for signal amplification. Applications include detecting autoantibodies associated with various diseases by looking for fluorescence patterns on tissue sections like Hep-2 cells or mouse organs. Indirect immunofluorescence is considered the standard technique as it has high sensitivity and specificity.
Immunohistochemistry (IHC) is a laboratory technique that uses antibodies to detect antigens in tissue samples. The document provides an overview of IHC, including its history, principles, steps, methods, applications and troubleshooting. Key developments include the indirect method in 1942 and enzyme conjugation in 1966. The main steps are tissue collection, fixation, sectioning, antigen retrieval, staining, detection and counterstaining. IHC is useful for cancer prognosis, infectious disease diagnosis and research applications by determining the presence or absence of cell markers.
Immunohistochemistry is a technique used to identify antigens in tissue samples using antigen-antibody interactions. It has made a large impact in disease diagnosis since its development in the 1940s-1970s. The technique involves using labeled antibodies that specifically bind to antigens in tissue sections. This binding is then visualized using markers like enzymes or fluorescent dyes. Several methods have been developed to increase the signal and reduce background noise, including indirect labeling techniques and polymer-based methods. Proper tissue processing and antibody selection are important for obtaining high quality results with immunohistochemistry.
Immunohistochemistry (IHC) is a highly sensitive method that allows the localization of antigen within a cell or a tissue with high resolution. The method is based on the use of a primary antibody that specifically binds to its complementary antigen. The bound antibody may then be visualized by a variety of methods such as colorimetric end points.
THEORIES OF STAINING Biological Staining
Structural Components (Nature) Of Stains
Staining Mechanism
Metachromasia
Types Of Staining
Staining of Paraffin Section
A stain is any colouring organic compound that combined with another substance imparts a colour to that substance.
The term ‘dye’ is used to refer to a colouring agent that is used for general purposes, whereas the term ‘stain’ is used to refer to that dye which is used for biological purposes.
The stains used for bacteria are aniline dyes they are derived from aniline (C6H5NH2).
The most commonly used aniline dyes are crystal violet, methylene blue, basic fuchsin, safranin, eosin, etc.FACTORS INFLUENCING METACHROMASIA
Immunohistochemistry is a technique that uses antibodies to identify antigens in cells of a tissue sample. It relies on the principle of antibodies binding specifically to antigens in cells. The primary antibody binds to the antigen of interest, while the secondary antibody is conjugated to an enzyme or fluorescent label for visualization. A chromogen is used to produce a colored precipitate that can be seen under the microscope. Immunohistochemistry has many applications in pathology for tumor diagnosis and classification by identifying cellular markers of differentiation. It allows identification of cell lineages and tumor types through characteristic protein expression patterns revealed by specific antibody staining.
This document provides an overview of cancer immunology, including how tumors are able to evade and enhance immune responses. It discusses the types of tumors of the immune system, such as lymphomas and leukemias, and how tumors enhance their growth by modulating antigens, expressing low levels of MHC molecules, and lacking costimulatory signals. It also describes tumor antigens, how the immune system responds to tumors through cells like NK cells and macrophages, and immunotherapy approaches to boost anti-tumor immunity.
Enzymes are proteins that catalyze chemical reactions without being changed chemically. They act as catalysts by lowering the activation energy of reactions and increasing their rate. Enzymes are usually named by adding "ase" to the substrate they act on and can be classified into six groups including oxidoreductases, transferases and hydrolases. Enzyme histochemistry is used in muscle disease diagnosis by demonstrating enzyme activity and localization in tissue sections.
The document discusses the history, utility, and methods of preparing cell blocks from fine needle aspiration cytology samples. Cell blocks allow examination of histological structure and use of ancillary tests. Key methods include the fixed sedimentation method using a 1:1 ratio of 100% alcohol and 40% formalin, the plasma thrombin method using equal parts plasma and thrombin, and the bacterial agar method using 3% agar. Cell blocks provide increased diagnostic sensitivity and specificity compared to cytology alone through examination of tissue architecture and ability to perform special stains and molecular testing.
This document provides an overview of immunohistochemistry (IHC), including its history, basic methods, considerations, and applications. IHC combines histology, immunology, and biochemical techniques to visualize the distribution and localization of specific cellular components within tissues through antigen-antibody reactions tagged with visible labels. Key steps discussed include fixation, sectioning, antigen retrieval, blocking, controls, direct and indirect antibody binding methods, detection methods including enzymatic and fluorescence techniques, and important considerations for optimizing IHC experiments.
Immunofluorescence is a technique that uses the binding of antibodies to antigens to detect the presence of substances. It relies on tagging antibodies with fluorescent dyes called fluorophores. When exposed to ultraviolet light, the fluorophore-tagged antibody complexes emit visible light that can be seen using a fluorescent microscope. There are several types of immunofluorescence assays including direct, indirect, and quantitative assays that are used to detect microorganisms, antibodies, and other biomolecules through the antigen-antibody reaction and fluorescent signal produced.
FIXATIVES in Pathology for Postgraduate and DMLTjenishJebadurai1
This document discusses various fixatives used in histology and cytology techniques. It begins by defining fixation as the process of preserving cells and tissue using physical or chemical methods. Commonly used fixatives include formaldehyde, glutaraldehyde, osmium tetroxide, mercury salts, picric acid, and alcohols. An ideal fixative would be non-toxic, low-cost, and effectively preserve tissue morphology, antigens, and nucleic acids while allowing for long-term storage. Factors like temperature, pH, concentration, and duration impact fixation quality. Proper fixation is important for minimizing artifacts and ensuring high quality staining.
The document discusses fixation in histology and cytology. It describes the aims of fixation as preserving tissue structure and preventing autolysis and bacterial growth. Fixation causes some changes like tissue shrinkage and hardening. The types of fixation include immersion, coating, vapor and perfusion. Formalin and alcohol are common fixatives. Factors like pH, temperature, duration and osmolarity influence fixation quality. The choice of fixative depends on the tissue and technique used, for example glutaraldehyde for electron microscopy. Useful fixative formulas are also provided.
Histological Techniques: Section 2:Fixation of tissuesMathew Joseph
Fixation is the process of preserving tissues and cells for examination by preventing autolysis and putrefaction. It aims to preserve tissues in a lifelike state as possible while allowing staining and sectioning. Formalin is the most common fixative due to its ability to crosslink proteins through methylene bridges and preserve morphology. Glutaraldehyde provides better ultrastructural preservation but is more expensive. Different fixatives are used depending on the goals, with alcohols and picric acid used for cytology and chromic acid and osmium tetroxide used for electron microscopy due to their effects on specific structures.
This document discusses tissue fixation in pathology. It begins by describing the overall tissue processing steps and importance of fixation. The main types of fixatives are then outlined, including coagulant, cross-linking, and compound fixatives. Formalin and glutaraldehyde fixation are discussed in depth. Key factors that influence fixation quality like buffer pH, fixation duration, tissue size, and temperature are also summarized. The document provides a comprehensive overview of fixation in pathology.
The document discusses fixation and processing of tissue specimens. It describes fixation as a process that preserves cells and tissues in a physical and chemical state to prevent biochemical changes and morphological distortion. The goals of fixation are to maintain the original tissue structure and prevent autolysis and bacterial growth. Common fixatives include formaldehyde and ethanol. The document outlines various types of fixatives and factors that influence fixation like buffer, pH, duration, temperature and concentration. It also discusses processing of fixed tissue, which provides rigidity for sectioning. Processing is influenced by viscosity, agitation, heat, vacuum and pressure. The document notes potential artifacts from improper fixation like formalin pigment or prolonged fixation effects.
This document provides an overview of tissue fixation. It begins with an introduction to fixation and its importance in histology. The main functions of fixatives are to prevent autolysis and putrefaction of tissue. Various fixation methods and types of fixatives are described, including aldehydes, metallic salts, picric acid, alcohols, chromates, and osmium tetroxide. The document discusses the reactions of fixatives with proteins, nucleic acids, lipids, and carbohydrates. Common fixatives like formaldehyde and glutaraldehyde are compared. Factors affecting fixation and the effects of fixation are also summarized.
This document discusses protein and peptide drug delivery systems. It begins by defining proteins and peptides, and describing their structures. It then discusses various challenges in delivering protein and peptide drugs, such as stability issues like denaturation, aggregation, oxidation, and proteolysis. It also categorizes different drug delivery routes like parenteral, pulmonary, transdermal, and oral. Finally, it provides examples of marketed protein and peptide drug formulations and discusses strategies to improve stability and delivery of these drugs.
The document discusses microbial enzymes. It begins by defining enzymes as biological catalysts produced by living organisms. It then discusses the history of enzymes, noting that Wilhelm Kühne first coined the term in 1877. The document outlines various sources of microbial enzymes from soil, food, and degrading areas. Common microorganisms that produce enzymes include bacteria, fungi, and actinomycetes. Examples are provided of specific enzymes produced by different microorganisms. The document then discusses methods for isolating and purifying enzymes, including precipitation, chromatography techniques, and affinity purification. It provides examples of enzyme purification strategies and industrial applications of enzymes in areas like baking, brewing, dairy, pharmaceuticals, and more.
The document discusses various components of typical cell culture media, including carbohydrates, amino acids, salts, buffers, vitamins and hormones, antibiotics, and serum. It describes the purposes and considerations for each component in maintaining optimal cell growth conditions and metabolism. Key factors include maintaining isotonicity, buffering pH, providing nutrients and growth factors, and preventing bacterial/fungal contamination.
This document provides guidance on grossing techniques for pathology specimens. It discusses proper specimen identification, labeling, collection, fixation and storage. The ideal properties of fixatives are outlined, including preventing autolysis and bacterial growth while maintaining tissue morphology. Common fixatives like neutral buffered formalin are described along with their advantages and limitations. The key steps of gross examination involve describing the location, size, shape and abnormalities seen in specimens before selecting portions for microscopic analysis.
This document discusses proper tissue fixation, which is important for immunohistochemistry (IHC). It begins by defining fixation as the process of preserving tissue using formalin solution. Formalin fixation occurs via a slow, two-step chemical reaction involving formaldehyde that crosslinks proteins over time. The rate of fixation depends on factors like tissue type and size. Proper fixation is crucial for IHC because it preserves tissue structure while crosslinking can mask antigens, requiring antigen retrieval techniques before IHC staining.
This document discusses tracing the evolution of the human body through analyzing the chemical evolution of proteins and other molecules in the body over time. It notes that tracking changes in conserved proteins that control fundamental processes, like the Pax6 gene which regulates eye development, can reveal how closely related different organisms are. It recommends using the human arrestin protein sequence as an example, performing BLAST searches to find the arrestin sequence in the human genome and other organism genomes, then aligning the protein sequences to compare percentages and determine evolutionary relationships.
Histochemistry in pathology and it'sbranches.pptxDrMUSTAFAAlAmeri
Thi is a simple overview about histochemistry and its branches, how the histological diseases can be diagnosed and what methods are used to examine the tissue.
This document discusses various techniques used in immunoblotting and blotting. It begins by defining blotting as techniques used to visualize specific DNA, RNA, and proteins among contaminants. It then describes three main types of blotting - western blotting for proteins, northern blotting for RNA, and southern blotting for DNA. The document focuses on western blotting and immunoblotting. It provides details on tissue preparation, gel electrophoresis, protein transfer, blocking, detection, analysis, and applications of western blotting and immunoblotting techniques.
The document discusses in situ gel drug delivery systems, which are liquid before administration but gel after contact with bodily fluids or tissues. It describes various polymers used to form in situ gels via temperature, pH, or ion triggers. It also categorizes in situ gel systems based on their gelation mechanism and administration route.
Histopathology examines minute tissue alterations from disease. Samples come from cadavers, autopsies, animal tissues, or biopsies. Histopathological examination is useful for establishing disease pathogenesis and diagnosing diseases that are difficult to diagnose by other means. It typically begins with surgery or biopsy to collect tissue samples, which are then fixed, processed, and examined microscopically. Common fixatives include formaldehyde and glutaraldehyde, which cross-link proteins to preserve tissue morphology and prevent autolysis.
Endotoxin Testing is performed to ensure that injectable preparations and medical devices are free from pyrogens and safe for human use.
Pyrogens constitute a heterogeneous group of fever causing substances which comprise both microbial and non-microbial substances. The most potent and most widely known are the endotoxins or lipopolysaccharides (LPS), which are cell wall components of gram-negative bacteria. Gram-positive bacteria are also sources of pyrogens, in particular lipoteichoic acid (LTA), as are particles from yeasts and viruses. Non-microbial pyrogens often emanate from production environments. Small particles of packaging materials are a typical example.
Fixatives used in tissue processing - Histopath techniques.Kaaviya Subramaniam
This document discusses various types of fixatives used to preserve cell and tissue structure. It begins by explaining how living cells require oxygen and nutrients from blood circulation, and will die when cut off from this supply. Fixation is needed to preserve tissues as they were in life for histological examination. Various fixatives are then described, including physical, chemical, simple, compound, dehydrant, coagulant, cross-linking, and osmium tetroxide fixatives. The ideal properties and mechanisms of several common fixatives like formaldehyde, glutaraldehyde, picric acid, acetic acid, and mercuric chloride are also summarized.
Gross Examination, Selection, Collection and Fixation of Specimen ghulam abbas
The document discusses the gross examination, selection, collection and fixation of specimens in pathology. It covers identifying and labeling specimens, performing a gross examination, selecting relevant portions for microscopic examination, and principles of proper fixation. Fixation preserves tissue morphology and prevents autolysis and contamination. Common fixatives include 10% neutral buffered formalin, Bouin's solution, B5, and Zenker's solution. Proper handling and fixation are important for accurate laboratory diagnosis.
Evaluation of protein and peptide formulations.pptxDivya Pushp
This document discusses stability testing and evaluation methods for protein formulations. Stability testing ensures products maintain specifications over shelf life under various storage conditions. Evaluations include bioassays to assess potency, which can be in vitro by monitoring cell responses to proteins or in vivo by monitoring animal pharmacological responses. Common evaluation methods are UV spectroscopy, Bradford assay, thermal analysis like DSC and TGA, and chromatography techniques like HPLC, ion exchange, and chromatofocusing.
The document discusses various hematological investigations and artifacts. It describes the process of a differential count, which involves examining a peripheral blood smear under a microscope to determine percentages of different white blood cells. The blood smear can also reveal abnormal red blood cell and white blood cell morphologies. The document then discusses the steps to make a good blood smear, including using a clean slide and proper angulation and force when spreading the blood. Potential artifacts from improper smear preparation or staining are also described. The document concludes with discussing various hematological tests including complete blood count, erythrocyte sedimentation rate, coagulation tests, and factors that could affect the results.
The document discusses various investigations and artifacts in hematology. It covers topics like complete blood count, hemogram tests, tests of hemostatic function, blood collection methods, and common errors in hematological tests. The complete blood count includes tests like hemoglobin concentration, total erythrocyte count, total leukocyte count, and blood film examination. Hemostatic function tests include bleeding time, clotting time, prothrombin time, and fibrinogen determination. Proper blood collection and anticoagulant use are important to avoid hemolysis and other errors in test results.
The document provides an overview of immunohistochemical (IHC) techniques. It discusses the basic principles of IHC, including antigen-antibody reactions and the use of primary and secondary antibodies. It also describes different IHC staining methods such as direct, indirect, and peroxidase-antiperoxidase methods. Key enzymes and chromogens used in IHC are discussed, as well as factors that influence antibody binding such as dilution, incubation time and temperature.
This document provides an overview of red lesions that can occur in the oral cavity. It discusses normal variations in oral mucosa color and various factors that can affect color. Red lesions are classified and several common types are described in detail, including traumatic erythematous macules, purpuric macules, inflammatory fibrous hyperplasia, nicotine stomatitis, erythroplakia, carcinoma, and candidiasis. Diagnostic features, histopathology, differential diagnoses, and management are covered for key red lesions. The document aims to guide clinicians in identifying and diagnosing different oral red lesions.
This document provides a review of midline diastemas. It discusses the prevalence and various etiological factors that can cause midline diastemas, such as an enlarged labial frenum, tooth size discrepancies, missing teeth, supernumerary teeth, oral habits, and certain medical conditions. The management involves orthodontic treatment to close the space as well as potentially frenectomy surgery. Retention is important after treatment to prevent relapse of the diastema.
This document reviews non-invasive techniques for age estimation using dental development and changes. It discusses:
1. Several non-invasive dental age estimation techniques including Schour and Masler's atlas method, Demirjian's scoring method, and Kvaal's radiographic method of measuring secondary dentin deposition.
2. Factors used for dental age estimation including tooth emergence, mineralization, root development, attrition, and changes in dental tissue composition.
3. Applications of dental age estimation in different age groups from prenatal to adults. The most accurate methods vary depending on the age group.
4. Comparison of different dental age estimation methods finding that methods combining multiple teeth and examiners tend
This document describes a case report of a solitary angiokeratoma lesion found on the tongue of a 38-year-old male patient. Solitary angiokeratomas of the oral mucosa are rare. The lesion was a well-circumscribed, dark brown growth on the dorsal surface of the tongue. Histopathological examination revealed numerous dilated blood vessels in the papillary dermis along with hyperkeratosis and acanthosis of the epithelium, consistent with angiokeratoma. Immunohistochemical staining was positive for CD34, confirming the lesion contained proliferating blood vessels. No other lesions were found on the patient's body. The lesion was completely excised with no recurrence after 6 months of follow up.
Hematoxylin and eosin staining is the most widely used histological staining technique. It uses hematoxylin, which stains cell nuclei blue, and eosin, which stains cytoplasm and connective tissue pink. The process involves dewaxing tissue sections, hydrating them, staining with hematoxylin, differentiating with acid alcohol, counterstaining with eosin, dehydrating, and mounting. Hematoxylin requires a mordant like aluminum or iron to stain tissues. Commonly used hematoxylins include Harris', Mayer's, Ehrlich's, and Delafield's.
H and E staining is most important part of the histopathological diagnosis, this presentation is to highlight some important basic concept of the Staining.
This document provides an overview of the histology of the major salivary glands, including the parotid, submandibular, and sublingual glands. It describes the secretory end pieces composed of serous and mucous cells, as well as the ductal system including intercalated, striated, and excretory ducts. The minor salivary glands are also briefly discussed. The roles of myoepithelial cells and the different cell types involved in saliva production are summarized.
Hematoxylin and eosin staining is a common histological technique that uses hematoxylin, which stains cell nuclei blue, and eosin, which stains cytoplasm and connective tissue pink. The document describes the full H&E staining procedure, including dewaxing, hydration, staining, differentiation, dehydration, clearing and mounting of tissue sections. It also discusses the principles and properties of hematoxylin, including how it is extracted from logwood and requires oxidation or "ripening" to become an effective nuclear stain. Commonly used hematoxylin formulations including Harris's, Mayer's, and Ehrlich's are compared.
This document provides an overview of hematoxylin and eosin staining. It discusses the theory behind staining, including how dyes interact with tissues through various bonding mechanisms. It also describes factors that influence staining results, such as rates of dye uptake and loss, binding site affinities, and tissue modification during fixation. The document highlights how hematoxylin and eosin work as the most commonly used routine stain in histopathology.
This document provides information about the anatomy and development of the major and minor salivary glands. It discusses the parotid gland, submandibular gland, sublingual gland, and minor salivary glands. For each gland it describes the location, structure, duct system, blood supply, nerve innervation, and other key details. It also covers the embryonic development of the salivary glands from the initial bud formation through branching and lumen development.
This document provides an overview of oral epithelium, including its structure and function. It discusses the four main layers of stratified squamous epithelium found in the oral cavity - stratum basale, stratum spinosum, stratum granulosum, and stratum corneum. It describes the cells found in each layer, such as stem cells, transit amplifying cells, and keratinocytes, and the changes they undergo during differentiation and keratinization. The document also discusses non-keratinocyte cells like melanocytes, Langerhans cells, and Merkel cells. It provides details on cytoskeletal components, basement membrane, and the specialized oral mucosas.
Epithelium is a tissue that covers external surfaces and lines internal cavities and tubes of the body. It has several key characteristics including being a continuous sheet of cells attached to a basement membrane and generally being avascular.
There are several types of epithelium classified by the number of cell layers - simple (one cell layer), pseudostratified (cells appear in multiple layers but are all attached to the basement membrane), and stratified (multiple cell layers). Epithelium also includes glandular epithelium specialized for secretion. The seminar covered the classification, structure, and functions of the main types of epithelium.
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Histololgy of Female Reproductive System.pptxAyeshaZaid1
Dive into an in-depth exploration of the histological structure of female reproductive system with this comprehensive lecture. Presented by Dr. Ayesha Irfan, Assistant Professor of Anatomy, this presentation covers the Gross anatomy and functional histology of the female reproductive organs. Ideal for students, educators, and anyone interested in medical science, this lecture provides clear explanations, detailed diagrams, and valuable insights into female reproductive system. Enhance your knowledge and understanding of this essential aspect of human biology.
Does Over-Masturbation Contribute to Chronic Prostatitis.pptxwalterHu5
In some case, your chronic prostatitis may be related to over-masturbation. Generally, natural medicine Diuretic and Anti-inflammatory Pill can help mee get a cure.
Adhd Medication Shortage Uk - trinexpharmacy.comreignlana06
The UK is currently facing a Adhd Medication Shortage Uk, which has left many patients and their families grappling with uncertainty and frustration. ADHD, or Attention Deficit Hyperactivity Disorder, is a chronic condition that requires consistent medication to manage effectively. This shortage has highlighted the critical role these medications play in the daily lives of those affected by ADHD. Contact : +1 (747) 209 – 3649 E-mail : sales@trinexpharmacy.com
Local Advanced Lung Cancer: Artificial Intelligence, Synergetics, Complex Sys...Oleg Kshivets
Overall life span (LS) was 1671.7±1721.6 days and cumulative 5YS reached 62.4%, 10 years – 50.4%, 20 years – 44.6%. 94 LCP lived more than 5 years without cancer (LS=2958.6±1723.6 days), 22 – more than 10 years (LS=5571±1841.8 days). 67 LCP died because of LC (LS=471.9±344 days). AT significantly improved 5YS (68% vs. 53.7%) (P=0.028 by log-rank test). Cox modeling displayed that 5YS of LCP significantly depended on: N0-N12, T3-4, blood cell circuit, cell ratio factors (ratio between cancer cells-CC and blood cells subpopulations), LC cell dynamics, recalcification time, heparin tolerance, prothrombin index, protein, AT, procedure type (P=0.000-0.031). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and N0-12 (rank=1), thrombocytes/CC (rank=2), segmented neutrophils/CC (3), eosinophils/CC (4), erythrocytes/CC (5), healthy cells/CC (6), lymphocytes/CC (7), stick neutrophils/CC (8), leucocytes/CC (9), monocytes/CC (10). Correct prediction of 5YS was 100% by neural networks computing (error=0.000; area under ROC curve=1.0).
ABDOMINAL TRAUMA in pediatrics part one.drhasanrajab
Abdominal trauma in pediatrics refers to injuries or damage to the abdominal organs in children. It can occur due to various causes such as falls, motor vehicle accidents, sports-related injuries, and physical abuse. Children are more vulnerable to abdominal trauma due to their unique anatomical and physiological characteristics. Signs and symptoms include abdominal pain, tenderness, distension, vomiting, and signs of shock. Diagnosis involves physical examination, imaging studies, and laboratory tests. Management depends on the severity and may involve conservative treatment or surgical intervention. Prevention is crucial in reducing the incidence of abdominal trauma in children.
- Video recording of this lecture in English language: https://youtu.be/kqbnxVAZs-0
- Video recording of this lecture in Arabic language: https://youtu.be/SINlygW1Mpc
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These lecture slides, by Dr Sidra Arshad, offer a quick overview of the physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar lead (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
6. Describe the flow of current around the heart during the cardiac cycle
7. Discuss the placement and polarity of the leads of electrocardiograph
8. Describe the normal electrocardiograms recorded from the limb leads and explain the physiological basis of the different records that are obtained
9. Define mean electrical vector (axis) of the heart and give the normal range
10. Define the mean QRS vector
11. Describe the axes of leads (hexagonal reference system)
12. Comprehend the vectorial analysis of the normal ECG
13. Determine the mean electrical axis of the ventricular QRS and appreciate the mean axis deviation
14. Explain the concepts of current of injury, J point, and their significance
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. Chapter 3, Cardiology Explained, https://www.ncbi.nlm.nih.gov/books/NBK2214/
7. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
1. 1
Presented by
Dr. Shrikant Sonune
Guided by
Dr Ashok Patil,
Dr Shilpa Kandalgaonkar,
Dr Mayur Chaudhary,
Dr Suyog Tupsakhare,
Dr Mahesh Gabhane.
Immunohistochemical
technique (part II)
4. • 1941: Coons et all. have demonsrated antigens on tissue
sections by using an antibody which was linked to an florescent
label.
• 1966: Nakane and Pierce as well as Avrameas and Uriel
reported the use of secondary antibody which was conjugated
with peroxidase enzyme.
• 1970 : Sternberger et al described the peroxidase-
antiperoxidase technique.
• 1971: Envall & Perlman reported the use of alkaline
phosphatase labeling
5. • 1975: Kohler & Milstein described a revolutionary
method for production of monoclonal antibodies
• 1976 : Huang et al described the use of trypsin
digestion on paraffin sections as a means of reveling
some antigens otherwise masked by formalin fixation &
paraffin processing.
• 1977:Heggness & Ash proposed the use of avidin –
biotin for immunofluorescence.
6. • 1979: Sternberger described the peroxidase-antiperoxidase
method.
• 1981: Hsu et all. have defined the method of avidin-biotin-
peroxidase complex which is abbreviated as ABC method.
• 1984 : Alkaline phosphatase anti alkaline phosphatase
technique using a red reaction product.
• 1993: Advent of one step system, based on a two layer
dextran polymer. Reported by Pluzek et al at UK pathological
society meeting.
7. 1994 : Norton reported that the stainless steel
pressure cooker was more efficient than the
microwave oven for retrieving some antigens & this
was conformed by Miller et al.
1998: Dako launched a new labeling system based
on the dextran polymer technology.
8. • Intercellular antigens,
• Cell surface antigens,
• Tissue antigen for
diagnosing autoimmune
diseases
• Protein hormones in
histopathological diagnosis
• Soluble antigens of the cell
• Diagnosis of the endocrine
tumors
• Small amounts of peptides
in endocrine or
neuroendocrine cells
• Immunodeposits
• Tumoral markers
• Tumor typing
9. 1. Fixation
Fresh unfixed, fixed, or formalin fixation and
paraffin embedding
2. Sectioning
Tissue
preparation
10. 1. Antigen retrieval
Proteolytic enzyme method and Heat-induced method
2. Inhibition of endogenous tissue components
3% H2O2
Pretreatment
11. Make a selection based on the type of specimen,
the primary antibody, the degree of sensitivity and
the processing time required.
Staining
12. The requirements of a fixative vary according to the
different techniques employed
13. Blood smears are preserved by air-drying.
The subsequent fixation method used depends upon the
staining technique.
Routine air-drying for 1–2 hours does not have a
deleterious effect on most antigens studied
immunocytochemically.
Routine air-drying for 1–2 hours does not have a
deleterious effect on most antigens studied
immunocytochemically.
14. Most cytology smears are immediately fixed in 95%
ethanol or are spray-fixed with a carbowax
containing alcoholic fluid.
Ethanol fixation prevents staining for most leukocyte
markers, such as T and B cell antigens.
15. Two preparations can be made,
one wet-fixed
one air-dried.
With wet-fixed smears, one of the main problems is
the loss of cells, particularly clumps, during the
Immunostaning incubations.
16. Cryostat sections give much better antigen
preservation than paraffin sections.
Additionally, fixative can be used with cryostat
sections,
Different and optimal fixative for each antigen,
17.
18. The most popular fixatives contain
Formalin (40% w/v formaldehyde in water),
A neutral salt to maintain tonicity
A buffering system to maintain pH.
Well tolerated by tissues
Have good penetration.
Generally formalin-based fixatives are excellent for
most Immunostaning procedure.
19. Formaldehyde fixes tissue by reacting primarily with
basic amino acids to form cross-linking “methylene
bridges.”
Relatively low permeability to macromolecules and that
the structures of intracytoplasmic proteins are not
significantly altered.
The great variation in time and conditions for fixation
that cause majority of problems in immunochemistry.
20. Many antigens can be demonstrated after the use of
appropriate pretreatment methods,
Such as proteolytic enzyme digestion and/or antigen
retrieval, particularly if polyclonal antisera are used.
21. If monoclonal antibodies are to be utilized on
formalin-fixed, paraffin-embedded tissue sections,
then
Does formaldehyde react with the epitope under
investigation?
Does it react with adjacent amino acids causing
conformational changes?
Does paraffin processing destroy the epitope under
investigation?
22. If there are conformational changes resulting from
the reaction of formaldehyde with amino acids
adjacent to the epitope,
These can often be reversed using proteolytic
enzyme digestion or antigen retrieval.
23. If there are conformational changes in the epitope
due to tissue processing, these are irreversible.
Many epitopes are sensitive to heat, and during the
paraffin-embedding step, tissues are heated to the
melting point of wax, usually between 50–60°C.
24. Saturated (1.2% w/v) aqueous picric acid 75 mL
Formalin (40% w/v formaldehyde) 25 mL
Glacial acetic acid 5 mL
A saturated solution (w/v) contains 1.17 g/100 mL
distilled water.
25. This fixative penetrates rapidly and fixes all tissues very
well,
Fixation time is 1–12 hours
Lipid-containing antigens may be affected.
Tissues fixed in Bouin’s solution must be washed in
70% ethanol to precipitate soluble picrates prior to
aqueous washes.
After cutting sections, the yellow color in the tissue can
be removed by treatment with 5% (w/v) sodium
thiosulfate, followed by a water wash.
26. To improve cytological preservation and minimize
distortion associated with formaldehyde-based
fixatives, mercuric chloride-based fixatives are used.
These fluids are generally poor penetrators and are
not well tolerated by the tissues.
27. Frequently, mercuric chloride-based fixatives are
used secondarily to formalin.
Tissues are initially fixed in formal saline or neutral
buffered formalin.
Tissues are then immersed in the mercuric chloride-
containing fluid for a further period of fixation.
29. The permeability of the tissue is greater
Coagulant fixatives,
Antibody penetration is better,
Resulting in a more intense Immunostaning.
These fixatives are also having additive effect.
30. B5 is widely advocated for the fixation of lymph node
Biopsies
B5 STOCK SOLUTION
Mercuric chloride 2 g
Sodium acetate 2.5 g
Distilled water 200 mL
B5 WORKING SOLUTION
B5 Stock solution 20 mL
Formalin (40% w/v formaldehyde) 2 mL
31. ZENKER’S STOCK
SOLUTION
Mercuric chloride 50 g
Potassium dichromate 25 g
Sodium sulfate 10 g
Distilled water to 1 liter
ZENKER’S WORKING
SOLUTION
Zenker’s stock solution 100
mL
Glacial acetic acid 5 mL
CLEARING SOLUTION A
Iodine 0.5 g
70% ethanol 100 mL
CLEARING SOLUTION B
Sodium thiosulfate 5% (w/v)
in distilled water
32. Morphological detail is generally well preserved,
Penetration is poor
Fixation time is 2–15 hours at room temperature,
Sections are washed in running water for at least 1
hour to remove potassium dichromate deposits.
33. The tissue is then dehydrated using clearing agent A,
helps to remove mercuric deposits prior to sectioning.
After cutting sections and collecting on clean glass
slides,
The tissue again incubated in clearing solution A for 1–2
minutes.
Sections are rinsed in water, and placed in clearing
solution for 1–2 minutes.
34. This fixative is becoming increasingly popular, partly
because it preserves membrane proteins.
Zinc chloride 500 g
Formalin (40% w/v formaldehyde) 3 L
Glacial acetic acid 19 mL
Distilled water 20 L
35. WORKING SOLUTION
3% (w/v) Paraformaldehyde 50 mL M
Disodium hydrogen orthophosphate 100 mL
Lysine 0.9 g
Sodium periodate 0.15 g
Adjust to pH 7.4.
36. First described by McLean and Nakane
Preserve the micro anatomical relationship of cells
and cytological details.
Since the periodate oxidizes sugars to produce
aldehydes which are cross linked with lysine.
The paraformaldehyde stabilizes proteins.
37. A recent modification to this solution involves the
addition of potassium dichromate (PLDP).
This chemical is added to preserve lipids and the
fixative should therefore preserve all protein,
carbohydrate and lipid antigenic determinants.
38. It must be noted, however, that since additive
compounds are formed, immunoreactivity may be
blocked.
Furthermore, cytological detail is not as good as
with other fluids.
39. Sainte-Marie first described the use of alcohol
fixation and paraffin processing for
immunocytochemistry.
Small pieces of tissue are fixed rapidly and show
good cytological preservation.
40. They are in some ways ideal fixatives for
immunocytochemistry.
Since alcohols are coagulant fluids and do not form
additive compounds,
They permit good antibody penetration and do not
block immunoreactive determinants.
41. Conformational changes can occur.
Alcohols precipitate carbohydrates
Hence, useful for surface membrane antigens,
which often display carbohydrate-containing
epitopes.
42. Applied to frozen sections or smears.
Proteolytic digestion or antigen retrieval is of no use
following alcohol fixation
Ethanol fixation results in destruction of the tissue
section or smear.
43. Acetone is an excellent preservative of immunoreactive
sites, leaving most sites intact.
But it is a very poor penetrator.
It is used only for smears and cryostat sections.
Fixation is not complete, and extended incubation in
buffers may result in chromatolysis and loss of
membranes.
44. The dilemma of trying to obtain good morphological
preservation while maintaining immunoreactivity is
even more apparent in ultra structural
immunochemical studies.
For routine electron microscopy, it is usual to
employ glutaraldehyde primary fixation followed by
post fixation in osmium tetra oxide.
45. This combination produces excellent ultra structural
detail with good preservation of membranes.
With immunochemistry, however, the combination of
glutaraldehyde and osmium tetra oxide is not
generally useful.
46. Pre treatment of ultrathin sections with hydrogen
peroxide or sodium metaperiodate to counteract the
deleterious effects of osmium.
The glutaraldehyde primary fixation is not suitable
for many antigens, at least when employing the
concentrations of glutaraldehyde used for routine
electron microscopy.
47. WORKING SOLUTION
0.2 M Phosphate buffer, pH 7.4 50 mL
8% (w/v) Paraformaldehyde 25 mL
25% (w/v) Glutaraldehyde 0.8 mL
Distilled water 24.2 mL
48. If the tissue is frozen
The sections may need to be used in
immunohistochemistry
Acetone fixed:
- precipitates proteins onto cell surface---may extract lipids
- is needed for many of the “CD” antibodies
Unfixed:
Advantage: antigens are unaltered
Disadvantage: sections may fall off slide during staining
Paraformaldehyde fixed:
- needs to be freshly made, or frozen soon after
49. - Deparaffinize ( remove the infiltrated paraffin wax, by using organic solvents)
- The section then needs to be rehydrated, by sequential immersion
in graded alcohols (100%, 70% , 50% and then PBS)
- The deparaffinized section may need to be treated to expose buried
antigenic epitopes with either proteases or by heating in low pH citrate
buffer , or high pH EDTA buffer (Antigen Retrieval)
If the tissue is paraffin embedded
50. The loss of immunoreactivity by many antigens
caused as a result of fixation in formalin.
Hence many challenges for doing IHC in such cases
51. Epitopes 2 types
Formalin resistant- even after routine formalin fixation
this type of epitopes remains unchanged
Formalin sensitive- after routine formalin fixation this
type of epitopes shows substantial changes. It can be
divided into
• Irreversible
• Reversible or “Masked”
Rescued by antigen retrieval
52. The first attempt to “improve” the immunoreactivity
of formalin-fixed tissue antigens was by use of
tryptic digestion prior to immunofluorescent staining.
Proteolytic digestion compensates for the
impermeable nature of cross linked fixatives and
allowing hidden determinants to be exposed.
53. The concept of recovering lost immunoreactivity
Through exposure to heat near the boiling point of
water
But ,went against the tenet of protecting proteins from
the denaturing effect of heat.
54. The principle of antigen retrieval relies on the
application of heat for varying lengths of time to
formalin fixed paraffin embedded (FFPE) tissue
sections in an aqueous medium.
After deparaffinizing and rehydrating the tissue
sections, the slides are immersed in an aqueous
solution commonly referred to as a “retrieval
solution”.
55. The following techniques can retrieve many masked
antigens in routinely processed tissue:
Proteolytic enzyme digestion
Microwave antigen retrieval
Microwave and trypsin antigen retrieval
Pressure cooker antigen retrieval.
56. Pre- treating formalin-fixed routinely processed
paraffin sections with proteolytic enzymes to
unmask certain antigenic determinants was
described by Huange et al. (1976), Curran &
Gregory (1977). and Mepham et al. (1979).
The most popular enzymes-Trypsin
-Protease
57. It is generally accepted that the digestion somehow breaks
down formalin cross linking.
Hence the antigenic sites for number of antibodies are
uncovered.
Pure trypsin has a very limited digestion effect on formalin
fixed paraffin section
Essential for the demonstration of Cytokeratin & CD3.
58. Pitfalls of proteolytic digestion .
Under digestion results in very little staining.
Over digestion can result in very sever tissue
damage.
Batches of trypsin varies even from same source.
59. Pitfalls of proteolytic digestion .
Many antigens do not retrieved .
Some even destroyed by proteolytic enzyme.
The specificity of some primary antigens altered.
Allergy to trypsin
60. Remains largely unknown
Heat is obviously of great importance in reversing the
damages caused by the fixation with formalin and
embedding in paraffin.
Some cross-links are reversible (Schiff bases), thus
restoring the immunochemical integrity of the protein,
while others are not (methylene bridges).
Heat-induced AR has been more successful than the
use of proteolytic enzymes
61. Microwave oven heating retrieves many antigens
though previously lost or destroyed by routine
histopathological method.
Dewaxed section of tissue is boiled with the various
solutions like m-citrate buffer pH 6.
The prerequisite for this technique is use of strong
adhesive such as Vectabond or amino
propyltriethoxysilane(APES).
62. Replacing microwave oven with pressure cooker.
Optimized microwave oven heating not produce
consistent result.
Comparatively this method is consistent & less time
consuming.
Also require the strong adhesive.
63. Reported by Sandison et al in 1994
Heat pretreatment increases the sensitivity of
sections to the subsequent proteolytic digestion.
Drastically reduces the trypsin time
64. Advantages
Dilution factor of primary antibodies are greatly
increase.
Labeling of other antigens
Disadvantages
Proteolytic enzymes can destroy the sections.
Staining distribution can be altered.
66. Enzymes are most widely used labels.
Chromogen using standard histochemical method
produce a stable colored reaction end product
suitable for light microscopy.
Horseradish peroxidase is most widely used
enzyme.
67. There are also several chromogens for demonstration
of peroxidase.
Diaminobenzedine(DAB)tertrahydrochloride- brown
3-amino-9-ethylcarbazole- red
4chloro-l-naphthol-blue
Hanker-Yates reagent-dark blue
Alpha-naphthol pyronin –redish purple.
68. Colloidal gold is most common metal tracer in use.
Pink in color when observed in transmitted light.
Silver participation reaction can be used to amplify
the visibility of gold conjugate thereby allowing the
use of smaller, more stable gold conjugate.
Colloidal gold has much wider usage with the
electron microscope.
71. Indicate that proper staining technique was achieved
Use the same control over time
• Follow staining consistency over time
• Familiarity with pattern
73. Internal control
• Built-in or intrinsic controls
• Target antigen within normal tissue elements in addition
to tissue elements being evaluated
• Can replace external positive control
74. Internal control examples:
• S-100 protein in both melanoma and normal tissue
• Desmin present in blood vessel musculature
75. Used to determine antibody specificity
Use tissue without antigen expression
May need to identify negative cells within tissue
76. The ideal negative control is omission of primary
antibody.
In absorption control the primary antibodies are used
but not in pure form.
Pre-absorption of primary antibody with purified antigen.
This type of control is used in characterization &
evaluation of new antibodies.
77. The bonding between the primary & secondary
antibody is prevented in indirect technique by use of
some control.
This type of control is know as blocking control.
78. Textbook of microbiology Anathnarayan.6th
edition
Theory & practices of histologycal
thecnique – bancroff 5th edition
Immunohistochemical Staining Methods
Fourth Edition – Dako.
Science of lab diagnosis by john croker .
2nd edition.