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.
Immunohistochemistry (IHC) is the localization of a known antigen in tissues by utilizing antibodies directed towards that (specific) antigen. In this presentation, we will introduce the procedure of IHC and the troubleshooting solutions.
Immunohistochemistry (IHC) is the localization of a known antigen in tissues by utilizing antibodies directed towards that (specific) antigen. In this presentation, we will introduce the procedure of IHC and the troubleshooting solutions.
Antibodies are compelling proteins that are essential to the immune system and extremely powerful in biotechnology applications; existing as major players in our defence against external agents (viruses, bacteria, etc.), they are also widely used as tools for research, diagnosis and treatments.
Hybridoma Technology ( Production , Purification , and Application ) Sakshi Ghasle
Hybridoma technology revolutionized the field of immunology by enabling the production of monoclonal antibodies with high specificity and affinity. This presentation delves into the principles of DNA hybridoma technology, highlighting its significance in antibody production, therapeutic applications, and biomedical research. Learn about the key steps involved in generating hybridomas, from immunization to antibody screening, and discover the potential of recombinant DNA techniques in enhancing antibody engineering. Whether you're a student, researcher, or industry professional, this overview will provide valuable insights into the innovative world of hybridoma technology."
Uncover the wide-ranging applications of monoclonal antibodies in areas such as cancer therapy, autoimmune diseases, infectious diseases, and beyond. Learn about the latest advancements in antibody engineering and the development of novel therapeutic modalities, including bispecific antibodies, antibody-drug conjugates, and immune checkpoint inhibitors.
Whether you're a seasoned researcher or a newcomer to the field, this SlideShare presentation serves as a valuable resource for understanding the principles, techniques, and applications of hybridoma technology in modern biomedicine. Join a journey through the fascinating world of monoclonal antibodies and the groundbreaking science behind their creation.
Unlock the potential of hybridoma technology and propel your research to new heights. Dive into this SlideShare presentation now and explore the limitless possibilities of monoclonal antibody production with hybridoma technology.
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.
In Situ Polymerase Chain Reaction (In situ PCR) is a powerful method that detects minute quantities of rare or single-copy number nucleic acid sequences in frozen or paraffin-embedded cells or tissue sections for the localization of those sequences within the cells. The principle of this method involves tissue fixing (to preserve the cell morphology) and subsequent treatment with proteolytic digestion (to provide access for the PCR reagents to the target DNA). The target sequences are amplified by those reagents and then detected by standard immunocytochemical protocols. In situ PCR combines the sensitivity of PCR or RT-PCR amplification along with the ability to perform morphological analysis on the same sample, and thus it is an attractive tool in diagnostic applications. One of the most prominent applications is the detection of infectious disease agents including HIV-1, HBV, HPV, HHV-6, CMV, and EBV.
Xmatrx Infinity is a fully automated molecular pathology workstation designed to accelerate life sciences research and drug discovery and development. It is an open system that allows simultaneous optimization of 40 assay parameters in a single run. The 40 independently thermal cyclable workstations enable any slide-based staining assays, including IHC, ISH, CISH, FISH, multiplexing and co-detection, special stain, in situ PCR and miRNA. The Infinity system adapts and completely automates any manual protocols such as denaturation, hybridization, stringency washes, counter stain and final coverslip to maximize testing capacity, minimize hands-on time and ensure consistent results every time.
microRNA for Clinical Research and Tumor AnalysisBioGenex
The discovery of microRNAs [miRNAs] has been one of the defining developments in cancer biology over the past decade. miRNAs are short, single stranded 20-22 nucleotide long, non-coding RNAs that regulate gene expression and have fundamental roles in Cancer growth and metastasis. miRNAs exert their function via base pairing with complementary mRNA molecules, resulting in gene silencing via transcriptional repression or target degradation. BioGenex solved the inherent difficulties in visualizing miRNAs in spatial context by using a propriety technology to synthesize modified, high-affinity oligonucleotides, labelling miRNA probes with multiple reporter molecules and developing a fully-integrated miRNA-ISH workflow solution allowing high throughput analysis of miRNA in the spatial context.
Pathology Special Stains for FFPE Tissue StainingBioGenex
Special stains refer to alternative staining techniques that are used when H&E stains do not provide all the cellular information required. These techniques use a variety of dyes and methods so that pathologists can visualize tissue morphology and detect the presence of particular cell types, structures or pathogens (e.g. bacteria).BioGenex offers broadest special stain menu anywhere (over 30 special stains), including:
Grocott’s Methenamine Silver (GMS) Stain
Reticulin Stains
Trichrome Stains
Giemsa Stain
Periodic Acid-Schiff (PAS) Stains
Hematoxylin and Eosin (H&E) staining is a routine staining technique that reveals exceptional detail of tissue structure and makeup of the cells. Stained cell structures (e.g. nucleus, cytoplasm, organelles, extra-cellular components) provide important information for tissue-based cancer diagnosis. Special stains refer to alternative staining techniques that are used when H&E stains do not provide all the cellular information required. These techniques use a variety of dyes and methods so that pathologists can visualize tissue morphology and detect the presence of particular cell types, structures or pathogens (e.g. bacteria). We have the broadest special stain† menu anywhere (over 30 special stains), including:
Grocott’s Methenamine Silver (GMS) Stain
Reticulin Stains
Trichrome Stains
Giemsa Stain
Periodic Acid-Schiff (PAS) Stains
For More information Contact Customer support at customer.service@biogenex.com or follow the link http://biogenex.com/us/applications/special-stains/special-stains-controls.html
Xmatrx ELITE is BioGenx's All-in-One and All-at-Once fully automated system that provides an adaptable workflow solution for efficiency, productivity and profitability. The system consists of platform technology that standardizes complete automation from microtome to microscope in three simple steps (load, click and view) for any slide-based assays such as immuno-histochemistry (IHC), in situ hybridization (ISH), fluorescence in situ hybridization (FISH), co-detection and multiplex applications (double and triple stains; IHC/ISH), in situ PCR, micro-RNA and special staining. Hence, Xmatrx ELITE maximizes testing capacity, minimizes hands-on time, reduces errors and produces consistent results.
For more information contact Customer support at customer.service@biogenex.com
Fluoresence In Situ Hybridization Workflow Solution - Xmatrix NANO AutomationBioGenex
BioGenex eFISHiency system is the only integrated FISH workflow solution in the market offering an affordable, yet fast and accurate FISH automation. This system reduces hands-on processing time to less than 30 minutes, has the ability to run up to 10 different FISH protocols simultaneously and provides validated reagents and protocols for optimal results.
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THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
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(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
3. Introduction
• Histochemistry is a science that combines the techniques of biochemistry and
histology in the study of the chemical constitution of tissues and cells.
• Immunology is a science that deals with the immune system, cell-mediated and
humoral aspects of immunity and immune responses.
• Immunohistochemistry (IHC) Immunohistochemistry is the localization of a known
antigen in tissues by utilizing antibodies directed towards that (specific) antigen.
3
6. Immunohistochemistry
Steps - Fixation
• Helps to prevent
• Elution
• Degradation
• Modification
• Preserves the position of the Ag
• Preserves the secondary and tertiary structure to a possible extent
• Provides target for Ab molecules
• Formaldehyde is the preferred fixative
• Most of the Ab available are optimized for use with formaldehyde
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7. Immunohistochemistry
Steps – Slide preparation
• 2-4 micron tissue sections are cut
onto slides
• Charged slides provide adhesion to
tissue sections
• The tissues are further adhered to
the slides by baking at 60oC
• Deparaffinization
• Tissue is treated in a series of
xylene and alcohol to remove
paraffin.
7
BioGenex
TheIHCIndia.
NM-123
Coloncarcinoma
20033/2007
paraffin wax coated slide
8. Immunohistochemistry
Steps – Antigen Retrieval
• Enables the partial reversal of
formaldehyde induced
confirmational change of Ags.
• Increases the accessibility of the Ab
to the Ag.
• Two methods:
• Heat
• Enzyme digestion
• Choice of Ag retrieval depends on
the Ag to be demonstrated.
• Heat Induced Epitope Retrieval
(HIER) is widely used.
8
BioGenex
TheIHCIndia.
NM-123
Coloncarcinoma
20033/2007
9. Immunohistochemistry
Pre-treatment: HIER
• Tissues sections are heated to app 1000C
• Achieved by
• Microwave oven
• Pressure cooker
• Vegetable steamers
• Water bath
• Automated Immunostainers
• The cooling of sections slowly allows the
protein to refold properly
• Protease Induced Epitope retrieval (PIER)
• Proteolytic enzymes cleave the protein to
release Antigenic sites
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10. Immunohistochemistry
Pre-treatment: Blocking
• Peroxide Block
• Blocks endogenous
peroxidases
• 3% H2O2
• Protein Block
• Blocks all non specific sites
• Reduces background
• 10% Normal serum is used
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BioGenex
TheIHCIndia.
NM-123
Coloncarcinoma
20033/2007
11. Immunohistochemistry
Primary Antibodies
Two types of Abs
• Polyclonal Abs:
• Produced by injecting an
animal with antigen and
harvesting the sera
• Monoclonal Abs :
• Produced by Hybridomas
11
BioGenex
TheIHCIndia.
NM-123
Coloncarcinoma
20033/2007
12. Immunohistochemistry
Direct Method
• Direct Method
• Labelled Ab reacts directly with
Ag in tissue sections
• Single step method
• Short and quick
• Insensitive due to little signal
amplification
• E.g., FITC conjugated Antisera
12
13. Immunohistochemistry
Indirect Method
• Unlabelled Primary Ab reacts with Ag and
the labelled secondary Ab reacts with the
primary Ab.
• Sensitive due to signal amplification
• Economical as single secondary Ab can be
used against many Abs from same species
• Peroxidase Anti-Peroxidase/ Alkaline
Phosphatase Anti-Alkaline
Phosphatase (PAP/ APAAP) Method
• Avidin-Biotin Complex (ABC) Method
• Streptavidin – Peroxidase Method
13
14. Immunohistochemistry
Detection Methods
• Ag-Ab conjugates are visualized by
the use of a label.
• Enzymes that produce a colored
precipitate in the presence of a
substrate are used as labels
• Labels :
• Peroxidase
• Alkaline Phosphatase
• Detection systems:
• Direct or Single step Method
• Indirect or Two step Method
14
BioGenex
TheIHCIndia.
NM-123
Coloncarcinoma
20033/2007
A B
15. Immunohistochemistry
Enzyme Labels
• Enzyme labels produce a colored
precipitate in the presence of a
specific substrate
• Most widely used label is Peroxidase
• Produces a dark brown precipitate
when Diamino Benzidine (DAB) is
added.
• Alkaline phosphatase is also used and
produces either red or blue
precipitates.
15
BioGenex
TheIHCIndia.
NM-123
Coloncarcinoma
20033/2007
16. Immunohistochemistry
Counter Staining
• Provides contrast to the primary
stain
• Most commonly used counter stain
is Hematoxylin and Eosin staining.
It is considered to be gold standard
in IHC
• Hematoxylin stains nucleic acids
blue while Eosin stains eisonophilic
structures in shades of red, pink
and orange.
16
SPECIMEN
TheIHCIndia.
NM-123
Coloncarcinoma
20033/2007
18. Immunohistochemistry
Controls
• Positive Controls:
• Cells or tissues that are known to contain the specific Ag
• Detects false negatives due to fixation and processing.
• It is used to validate the protocol or procedure used
• Negative Controls:
• Omission of Primary Ab with the same tissue and procedure
• Useful to detect endogenous biotin and peroxidase activity
18
19. Immunohistochemistry
Automation
• Fully automated IHC work stations are a
common practice
• Advantages:
• Greater consistency of staining
• Fast and accurate results
• Decreased use of reagents
• Less use of man power
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21. Immunohistochemistry
Troubleshooting: Weak or No staining
21
Sources Solutions
Inadequate deparaffinization
Deparaffinize sections longer or change
fresh xylene
Inactive primary antibodies Replace with a new batch of antibodies
Antibodies do not work due to improper
storage
Aliquot antibodies into smaller volumes and
store in freezer (-20 to -70℃) and avoid
repeated freeze and thaw cycles.
Antibody concentration was too low
Increase the concentration of antibodies. Or
run a serial dilution test to determine the
optimal dilution that gives the best signal to
noise ratio
Inadequate antibody incubation time Increase antibody incubation time
Inadequate or improper tissue fixation
Increase duration of post fixation or try
different fixatives
22. Immunohistochemistry
Troubleshooting: Weak or No staining
22
Sources Solutions
Tissue over-fixation
Reduce the duration of post-fixation or
perform an appropriate antigen retrieval
procedure
Incompatible secondary and primary
antibodies
Use secondary antibody that will interact
with primary antibody.
Inactive secondary antibody or other
reagents
Replace with a new batch of reagents
Inadequate substrate incubation time Increase the substrate incubation time
Incorrect mounting medium Choose a correct mounting medium
Reagents applied in wrong order or steps
omitted
Check notes or procedure used
23. Immunohistochemistry
Troubleshooting: Over staining
23
Sources Solutions
The concentration of antibodies was
too high
Reduce antibody concentration or perform
a titration to determine the optimal
dilution for primary and secondary
antibodies
Incubation time was too long Reduce incubation time
Incubation temperature was too high Reduce incubation temperature
Substrate incubation time was too long Reduce substrate incubation time
Sections dried out Avoid sections being dried out
24. Immunohistochemistry
Troubleshooting: High Background
24
Sources Solutions
The concentration of antibodies was
too high
Reduce antibody concentration or
perform a titration to determine the
optimal dilution for primary and
secondary antibodies
Incubation time was too long Reduce incubation time
Incubation temperature was too high Reduce incubation temperature
Substrate incubation time was too
long
Reduce substrate incubation time
Sections dried out Avoid sections being dried out
25. Immunohistochemistry
Applications
• Tumor Pathology
• Classification of Neoplasma
• Diagnosis of Malignancy
• Prognostic Markers
• Predicting response to treatment
• Detection of metastases
• Screening of inherited cancer syndromes
• Non- Tumor Pathology
• Neurodegenerative diseases
• Brain trauma
• Muscle diseases
• Amyloidosis
• Dementias
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