This document provides an overview of immunohistochemistry methods. It defines key terms like antigens, antibodies, affinity, and sensitivity. It discusses the history of immunohistochemistry from the 1930s to current techniques. The principles of immunohistochemistry are described, including production of primary reagents, tissue fixation, antigen retrieval, staining, and limitations. Various immunohistochemistry methods are summarized such as direct, indirect, polymer, peroxidase-antiperoxidase, and alkaline phosphatase techniques.
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 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.
Immunofluorescence : Immunofluorescence is a powerful technique that utilizes fluorescent-labeled antibodies to detect specific target antigens..
Fluorescein is a dye which emits greenish fluorescence under UV light. It can be tagged to immunoglobulin molecules.
This technique is sometimes used to make viral plaques more readily visible to the human eye.
Immunofluorescent labeled tissue sections are studied using a fluorescence microscope.
This method is used to visualise the localisation and quantity of a protein of interest. The target protein is bound to by a specific primary antibody, which in turn is detected by a secondary antibody conjugated to a fluorophore. A fluorescent or confocal microscope is used to visualise the protein.
Immunocytochemistry (ICC) differs from immunohistochemistry (IHC) in that the former is performed on samples of intact cells that have had most, if not all, of their surrounding extracellular matrix removed. In contrast, immunohistochemical samples are sections of biological tissue, where each cell is surrounded by tissue architecture and other cells normally found in the intact tissue. These differences cause the samples to be prepared differently. For ICC, the sample requires permeabilisation so that the antibodies can reach the intracellular targets. Depending on the thickness of the sample, IHC samples do not require this.
Do you have a technical question? Get in touch: info@stjohnslabs.com
cytology of urine tract - this slide contains the specimen collection method, preparation of specimen, types of fixatives, other preparation techniques, urinary tract histology, normal urinary tract cytology,
Immunofluorescence : Immunofluorescence is a powerful technique that utilizes fluorescent-labeled antibodies to detect specific target antigens..
Fluorescein is a dye which emits greenish fluorescence under UV light. It can be tagged to immunoglobulin molecules.
This technique is sometimes used to make viral plaques more readily visible to the human eye.
Immunofluorescent labeled tissue sections are studied using a fluorescence microscope.
This method is used to visualise the localisation and quantity of a protein of interest. The target protein is bound to by a specific primary antibody, which in turn is detected by a secondary antibody conjugated to a fluorophore. A fluorescent or confocal microscope is used to visualise the protein.
Immunocytochemistry (ICC) differs from immunohistochemistry (IHC) in that the former is performed on samples of intact cells that have had most, if not all, of their surrounding extracellular matrix removed. In contrast, immunohistochemical samples are sections of biological tissue, where each cell is surrounded by tissue architecture and other cells normally found in the intact tissue. These differences cause the samples to be prepared differently. For ICC, the sample requires permeabilisation so that the antibodies can reach the intracellular targets. Depending on the thickness of the sample, IHC samples do not require this.
Do you have a technical question? Get in touch: info@stjohnslabs.com
cytology of urine tract - this slide contains the specimen collection method, preparation of specimen, types of fixatives, other preparation techniques, urinary tract histology, normal urinary tract cytology,
General principle of immunoassay Theoretical basis and optimization of immun...Ashish Gadage
Unlock the mysteries of immunoassays with this comprehensive PowerPoint presentation. Delve into the fundamental principles that underpin immunoassay techniques, exploring the theoretical foundations and key concepts. From antigen-antibody interactions to signal amplification strategies, this presentation provides valuable insights into the world of immunoassay science.
Key Topics:
Basics of Immunoassay: Antigen-Antibody Interactions
Types of Immunoassays: ELISA, Western Blot, and More
Signal Detection and Amplification Techniques
Factors Affecting Assay Sensitivity and Specificity
Optimization Strategies for Enhanced Performance
Emerging Trends in Immunoassay Technology
Who Should View:
Designed for scientists, researchers, and students in the fields of immunology, biochemistry, and medical diagnostics. Whether you're new to immunoassays or seeking advanced insights, this presentation caters to a broad audience.
Presenter: Mr. Gadage Ashish Rambhau
(M Pharm Pharmacology)
Pravara Rural Education Society pravaranagar,Loni .
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.
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Immunohistochemistry is one of the most advance techniques in immuno-staining. In this presentation is a brief introduction to what is IHC, the principle, procedure and applications.
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Ve...kevinkariuki227
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
These lecture slides, by Dr Sidra Arshad, offer a quick overview of 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 leads (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
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. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
- Video recording of this lecture in English language: https://youtu.be/lK81BzxMqdo
- Video recording of this lecture in Arabic language: https://youtu.be/Ve4P0COk9OI
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
- Link to NephroTube website: www.NephroTube.com
- Link to NephroTube social media accounts: https://nephrotube.blogspot.com/p/join-nephrotube-on-social-media.html
Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
Dr. Vidisha Kumari, a leading epidemiologist in Bangalore, emphasizes the importance of getting vaccinated. "The flu vaccine is our best defense against the influenza virus. It not only protects individuals but also helps prevent the spread of the virus in our communities," he says.
This year, the flu season is expected to coincide with a potential increase in other respiratory illnesses. The Karnataka Health Department has launched an awareness campaign highlighting the significance of flu vaccinations. They have set up multiple vaccination centers across Bangalore, making it convenient for residents to receive their shots.
To encourage widespread vaccination, the government is also collaborating with local schools, workplaces, and community centers to facilitate vaccination drives. Special attention is being given to ensuring that the vaccine is accessible to all, including marginalized communities who may have limited access to healthcare.
Residents are reminded that the flu vaccine is safe and effective. Common side effects are mild and may include soreness at the injection site, mild fever, or muscle aches. These side effects are generally short-lived and far less severe than the flu itself.
Healthcare providers are also stressing the importance of continuing COVID-19 precautions. Wearing masks, practicing good hand hygiene, and maintaining social distancing are still crucial, especially in crowded places.
Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
2. Contents
• Introduction
• Definitions
• History
• Principle
• Production of primary reagents
• Technique
• Immonohistochemistry methods
• Tissue fixation
• Unmasking of antigen sites
• Immunohistochemistry in practice
• Counterstaining
• Limitations
• current application
3. Introduction
• Immunohistochemistry is as the name implies, a combination of two
disciplines – immunology and histology. The Immunohistochemistry
technique is used not only to determine if a tissue express or does not
express a particular antigen but also to determine the antigenic status of
particular cells within that tissue and microantomic location of the
antigen.
• Immunohistochemistry uses antibodies to distinguish antigenic
difference between the cells
4. Definitions
Immunohistochemistry
• This is a technique for identifying cellular or tissue constituents
(antigens) by means of antigen antibody interactions, the site of
antibody binding being identified either by direct labeling of the
antibody, or by use of a secondary labeling method.
• Immunohistochemistry- using tissue sections.
• Immunocytochemistry – cytological preparations
5. Antigens
• An antigen is a molecule that induces the formation of an antibody and
bears one or more antibody binding sites. These are highly specific
topographical regions composed of a small number of amino acids or
monosaccharide units, being known as antigenic determinant groups or
epitopes.
• The outer surfaces of antigens are covered by unique 3- dimensional
protein structures known as epitopes
6. Antibody
• Antibodies belong to the class of serum proteins known as immunoglobulins.
The terms antibody and immunoglobulin are often used interchangeably. They
are found in blood and tissue fluids, as well as many secretions.
• The basic unit of each antibody is a monomer. An antibody can be monomeric,
dimeric, trimeric, tetrameric, or pentameric. The monomer is composed of two
heavy and two light chains.
• There are five types of antibody found in the blood of higher vertebrates: IgA,
IgD, IgE, IgG, and IgM. IgG is the commonest and the most frequently used
antibody for immunohistochemistry.
7. Antibody-antigen binding
• The associated antibody and antigen are held together by a
combination of hydrogen bonds, electrostatic interactions and
van der Waals’ forces.
8. Affinity
• Affinity is the three-dimensional fit of the antibody to its specific antigen
and is a measure of the binding strength between the antigenic epitope and
its specific antibody-combining site.
Avidity
• Avidity is a related property referring to the heterogeneity of the
antiserum which will contain various antibodies reacting with different
epitopes of the antigen molecule. Avidity therefore is the functional
combining strength of an antibody with its antigen.
9. Antibody specificity
• This refers to the characteristics of an antibody to bind selectively to a
single epitope on an antigen
Sensitivity
• This refers to the relative amount of antigen that an
immunohistochemical technique is able to detect.
10. History
• The principle has existed since the 1930s.
• Started in 1941 when Albert H. Coons et al tagged fluorescein dye to
antibodies against pneumococci.
• Nakane and pierce et al. (1967) introduced enzyme peroxidase enabling IHC
staining to be viewed with light microscope.
• Colloidal gold label (Faulk andTaylor 1971) has also been discovered and used
to identify immunohistochemical reactions at both light and electron
microscopy level.
• Mason and Sammons in 1978 introduced alkaline phosphatase.
• In 1979, Sternberger described the peroxidase-antiperoxidase (PAP) method.
11. Principle of Immunohistochemistry:
• Immunohistochemistry is a method for localizing specific antigen in
tissues or cells based on antigen antibody reaction.
• The site of antibody binding is identified either by tagging the antibody,
directly or indirectly with a visible label.
• Fluorescent dye, colloidal metal, hapten, radioactive marker.
12. Production of primary reagents
• It is the pivotal reagent common all IHC techniques.
• They impart specificty.
• Two types of antibodies are used:
• Polyclonal antibodies.
• Monoclonal antibodies.
13. Polyclonal antibodies
• They are a heterogenous mixture of antibodies directed against various epitopes
of same antigen.
• Generated by different B-cell clones of the animals → immunochemically
dissimilar.
PROCEDURE:
• Produced by immunizing an animal with a purified specific molecule
(immunogen) bearing the antigen of interest.
• The animal will mount a humoral response to the immunogen and the
antibodies so produced can be harvested by bleeding the animal to obtain
immunoglobulin-rich serum.
• It is likely that the animal will produce numerous clones of activated plasma
cells (polyclonal). A polyclonal antiserum is therefore a mixture of antibodies
to different epitopes on the immunogen.
14.
15. Monoclonal antibodies
• They are a homogenous population of Ig directed against a single epitope.
• Generated by a single B-cell clone from one animal → immunochemically
similar.
• Developed by Kohler and Milstein in 1975.
PROCEDURE:
• The method combines the ability of a plasma cell or transformed B
lymphocyte to produce a specific antibody with the in vitro immortality of a
neoplastic myeloma cell line..
• With the technique of cloning, this cell can be grown and multiplied in cell
culture or ascetic fluid, theoretically to unlimited numbers.
• The result is a constant, reliable supply of one pure antibody with known
specificity.
16.
17. MONOCLONAL POLYCLONAL
Mouse or rabbit hybridoma Many different species (mostly rabbits)
Tends to be cleaner Tends to have more non-specific reactivity
More likely to get false- negative results if
target epitope is damaged or altered
More likely to have success in an unknown
application
Expensive to produce Inexpensive
Training is required for the technology
used
Skills required are low
Time scale is long for hybridomas Time scale is short
Recognizes only one epitope on an
antigen
Recognizes multiple epitopes on ant one
antigen
Can produce large amount of specific
antibodies
Produces large amounts of non specific
antibodies.
18. Labels
1) Enzyme labels
• Enzymes are the most widely used labels in immunohistochemistry, and
incubation with a chromogen using a standard histochemical method
produces a stable, colored reaction end-product suitable for the light
microscope
• Horseradish peroxidase is the most widely used enzyme, and in
combination with the most favored chromogen, i.e. 3,3α-
diaminobenzidene tetrahydrochloride (DAB).
19. • Horseradish peroxidase is commonly used as an antibody label for
several reasons:
Its small size does not hinder the binding of antibodies to adjacent sites.
Chance of contamination is minimized.
Stable enzyme.
Endogenous activity is easily quenched.
20. • Calf intestinal alkaline phosphatase is the most widely used
alternative enzyme tracer to horseradish peroxidase, particularly since
the development of the alkaline phosphatase-anti-alkaline phosphatase
(APAAP) method in 1984 by Cordell et al.
• Bacterial-derived β-D-galactosidase has also been used as a tracer
21. 2) Colloidal metal labels
• When used alone, colloidal gold conjugates appear pink when viewed
using the light microscope. An silver precipitation reaction can be used to
amplify the visibility of the gold conjugates.
3) Fluorescent labels
4) Radiolabels
22. Chromogens
• 3,3α-diaminobenzidene tetrahydrochloride (DAB), it yields a crisp,
insoluble, stable, dark brown reaction end-product. Although DAB has been
reported to be a potential carcinogen, the risk is now thought to be low.
• 3-amino-9-ethylcarbazole -red
• 4-chloro-1-naphthol - blue;
• Hanker-Yates reagent - dark blue
• α-naphthol pyronin - red-purple
24. Immunohistochemical methods
Traditional direct technique
• The primary antibody is conjugated directly to the label. The conjugate
may be either a fluorochrome (more commonly) or an enzyme. The
labeled antibody reacts directly with the antigen in the histological or
cytological preparation.
• Quick and easy to use.
• Provides little signal amplification
• Lacks the sensitivity.
25. New Direct technique
• Pluzek et al in 1993
• Commercial name- Enhanced Polymer One-step Staining (EPOS)
• A large number of primary antibody molecules and peroxidase enzymes
are attached to a dextran polymer ‘backbone’, hence increasing the signal
amplification and provide greater sensitivity.
26. Two-step indirect technique
• A labeled secondary antibody directed against the immunoglobulin of
the animal species in which the primary antibody has been raised
visualizes an unlabeled primary antibody.
• Horseradish peroxidase labeling is most commonly used, together with
an appropriate chromogen substrate.
• More sensitive technique because multiple secondary antibodies may
react with different antigenic sites on the primary antibody, thereby
increasing the signal amplification.
27. Polymer chain two-step indirect technique
• This technology uses an unconjugated primary antibody, followed by a
secondary antibody conjugated to an enzyme (horseradish peroxidase)
labeled polymer (dextran) chain.
• Conjugation of both anti-mouse and anti-rabbit secondary antibodies
enables the same reagent to be used for both monoclonal (rabbit and
mouse) and polyclonal (rabbit) primary antibodies.
28. Unlabeled Antibody Methods
PEROXIDASE ANTIPEROXIDASE METHOD / PAP
• Immune complex typically consists of 2 antibody molecules and 3 HRP
molecules in the configuration.
• The PAP reagent and the primary antibody must be from the same species,
whereas the bridge or linking antibody is derived from a second species and
has specificity against the primary antibody and the immunoglobulin
incorporated into the PAP complex.
29. ALKALINE PHOSPHATASE–ANTIALKALINE PHOSPHATASE
METHOD / APAAP
• Principle same as those described for the PAP method except that the PAP
complex is replaced with an APAAP complex.
The method has had three major
applications:
(1)staining of tissues with high levels of
endogenous peroxidase,
(2)double immunostaining in conjunction
with peroxidase,
(3)staining of specific cell types that benefit
from the bright red color of alkaline
phosphatase substrates
30. Immunogold silver staining technique (IGSS)
• Introduced by Faulk andTaylor (1971).
• In this method the gold particles are enhanced by the addition of metallic
silver layers to produce a metallic silver precipitate which overlays the
colloidal gold marker.
• silver lactate as the ion supplier
• hydroquinone as the reducing agent.
• Disadvantage: Formation of fine silver deposits in the background
31. Biotin-Avidin Procedure
• The biotin-avidin procedure exploits the high affinity binding between
biotin and avidin.
Biotin is linked chemically to the primary antibody,
Produces biotinylated conjugate that localizes to the sites of antigen
Avidin which is chemically conjugated to horseradish peroxidase, is added;
avidin binds tightly to the biotinylated antibody, thus
localizing the peroxidase moiety at the site of antigen in the tissue section.
32. Disadvantage:
1) Different batches of biotin and different batches of avidin have
differing affinities for one other → affects the sensitivity
2) Produces non- specific (false-positive) staining.
Advantage: Rapid
33. (Strept) avidin-biotin techniques
• The labeled streptavidin-biotin technique is the most widely used
methodology in diagnostic immunohistochemistry.
• 3 -step technique:
1. unconjugated primary antibody as the first layer,
2. followed by a biotinylated secondary antibody.
3. The third layer is either a complex of enzyme-labeled biotin and
streptavidin, or enzyme-labeled streptavidin
The enzyme can be either horseradish peroxidase or alkaline phosphatase,
used with a chromogen of choice
34. • Streptavidin has now largely replaced the use of avidin in
immunohistochemical detection techniques.
35. Biotinylated tyramide signal amplification
• Bobrow et al. first described the use of biotinylated tyramide to enhance
signal amplification, in 1989.
• The technique is based around the streptavidinbiotin technique.
• Advantage: Enables many antigens which had previously been unreactive
in formalin-fixed paraffin-embedded tissue to be demonstrated.
• Disadvantage: Excessive background staining
36. Procedure:
Application of the primary antibody
subsequent incubations in biotinylated secondary
antibody
horseradish peroxidase-labeled streptavidin
subsequent treatment with the biotinylated tyramide
amplification reagent.
hydrogen peroxide
free biotin radicals.
These reactive biotin molecules bind covalently to
proteins adjacent to the site of the reaction.
37. Tissue fixation
• A prerequisite for all routine histological and cytological investigations is
to ensure preservation of tissue architecture and cell morphology by
adequate and appropriate fixation.
• The fixative should preserve antigenic integrity and should limit
extraction, diffusion, or displacement of antigen during subsequent
processing.
• Fixation prevents the autolysis and necrosis of excised tissues, enhances
the refractive index of tissue constituents and increases the resistance of
cellular elements to tissue processing.
38. • Show good preservation of morphologic details after embedding in a
support medium (e.g., paraffin).
• Good fixation is the delicate balance between under-fixation and over-
fixation.
• Ideal fixation is the balance between good morphology and good
antigenicity.
• Prompt fixation is essential to achieve consistent results.
• Poor fixation or delay in fixation causes loss of antigenicity or diffusion of
antigens into the surrounding tissue.
39. • Fixative can cause changes in the steric configuration of proteins, which
may mask antigenic sites (epitopes) and adversely affect binding with
antibody.
• It is well recognized that cross-linking fixatives (formaldehyde) alter the IHC
results for a significant number of antigens, whereas coagulant fixatives,
especially ethanol, have been reported to produce fewer changes, although
there remains some controversy.
• A robust and optimized fixation protocol is a critical step in an
immunohistochemistry protocol as an antigen that has been inappropriately
fixed may not be detected in downstream detection.
40. • The most popular choice of fixatives for routine histology are formalin
based, either as a 10% solution or with the addition of different chemical
constituents.
• When formalin-based fixatives are used, intermolecular and intramolecular
cross-linkages are formed with certain structural proteins. These are
responsible for the masking of the tissue antigens.
• Methylene bridges forms between reactive sites on tissue proteins.
41. Advantages of formalin:
• good preservation of morphology, even after prolonged fixation
• economical chemical
• sterilizes tissue specimens in a more reliable way than precipitating
fixatives, particularly for viruses.
• Carbohydrate antigens are well preserved
• Cross-linking of protein in situ avoids leaching out of proteins that may
diffuse in water or alcohol.
• Many low–molecular-weight antigens (peptides) are well preserved in tissue
by formalin.
42. Unmasking of antigen sites
Manual methods for antigen unmasking include:
• Proteolytic enzyme digestion
• Microwave oven irradiation
• Combined microwave oven irradiation and proteolytic enzyme digestion
• Pressure cooker heating
• Decloaker heating
• Pressure cooker inside a microwave oven
• Autoclave heating
• Water bath heating
• Steamer heating
Before antigen unmasking pretreatments are employed, the sections are
dewaxed, rinsed in alcohol, and washed in water.
43. Proteolytic enzyme digestion
• Described by Huang et al. (1976), Curran and Gregory (1977), and
Mepham et al. (1979).
• The most popular enzymes employed today are trypsin and protease, but
other proteolytic enzymes such as chymotrypsin, pronase, proteinase K,
and pepsin may also be used.
• Principle- Digestion breaks down formalin cross-linking and hence the
antigenic sites for a number of antibodies are uncovered.
• Under-digestion results in too little staining, because the antigens are not
fully exposed.
• Over-digestion can produce false positive staining, high background
levels, and tissue damage.
44. Heat-mediated antigen retrieval techniques
• Heat-based antigen retrieval methods have brought a great improvement in
the quality and reproducibility of immunohistochemistry.They have also
widened its use as an important diagnostic tool in histopathology.
Theories:
Heavy metal salts (as described by Shi et al. 1991) act as a protein
precipitant, forming insoluble complexes with polypeptides, and
that protein precipitating fixatives display better preservation of
antigens than do cross-linking aldehyde fixatives
45. During formalin fixation intermolecular and methylene bridges and weak
Schiff bases form intramolecular cross-linkages, which may prevent it from
being recognized by a specific antibody.
Heat-mediated antigen retrieval removes the weaker Schiff bases but does
not affect the methylene bridges, so the resulting protein conformation is
intermediate between fixed and unfixed.
Morgan et al. (1997), who postulated that calcium coordination complexes
formed during formalin fixation prevent antibodies from combining with
epitopes on tissue-bound antigens.
High temperature weakens or breaks some of the calcium coordinate bonds,
but the effect is reversible on cooling
46. Microwave antigen retrieval
• Shi et al. (1991) first established the use of microwave heating for antigen
retrieval.
• Gerdes et al. (1992) used microwave antigen retrieval with a non-toxic
citrate buffer at pH 6.0 .
• Cattoretti et al. (1993) established microwave oven heating as an alternative
to proteolytic enzyme digestion.
• Antigen retrieval solutions: 0.01 M citrate buffer at pH 6.0 and 0.1 mM EDTA
at pH 8.0
• Uneven heating and the production of hot spots
47. Pressure cooker antigen retrieval
• Norton et al. (1994) suggested the use of the pressure cooker as an
alternative to the microwave oven. Batch variation and production of hot
and cold spots in the microwave oven could be overcome.
• Pressure cooking is said to be more uniform than other heating methods.
• A pressure cooker at 15 psi (10.3 kPa) reaches a temperature of around
120°C at full pressure
• It is preferable to use a stainless steel domestic pressure cooker, because
aluminum pressure cookers are susceptible to corrosion from some of the
antigen retrieval buffers
48. Steamer
• Steam heating appears to be less efficient than either microwave oven
heating or pressure cooking
• Advantage - less damaging to tissues than the other heating methods
49. Water bath
• Kawai et al. (1994) demonstrated that a water bath set at 90°C was
adequate for antigen retrieval.
• Increasing the temperature to 95–98°C, antigen retrieval was improved and
the incubation times could be decreased.
Advantage - gentler on the tissue sections because the temperature is set
below boiling point.
• antigen retrieval buffer does not evaporate and
• expensive commercial antigen retrieval solutions can be safely reused
Disadvantage -antigen retrieval times are increased compared to other
methods.
50. Combined microwave antigen retrieval and trypsin digestion
• Infrequently used today;
• Brief proteolytic digestion can be carried out before or after microwave
irradiation.
Advantages of heat pretreatment
• Some antigens previously thought lost in routinely processed paraffin-
embedded sections are now recovered by heat pretreatment.
• Many antigens are retrieved by uniform heating times, regardless of
length of fixation.
51. Pitfalls of heat pretreatment
• Care should be taken not to allow the sections to dry after heating, as
this destroys antigenicity.
• The boiling of poorly fixed material also damages nuclear detail.
• Fibrous and fatty tissues tend to detach from the slide.
Prevention:
• Vectabond or APES-coated slides (3-Aminopropylenetriethoxysilane)
can be dipped in 10% formal saline for 1–2 minutes and air dried before
picking up the sections.
52. Commercial antigen retrieval solutions
• There are numerous commercial antigen retrieval solutions available.They
can be either specialized high pH solutions (recommended for certain
antibodies) or lower pH 6.0 for more general use.
• Citrate buffer pH 6.0
• EDTA buffer pH 8.5
• Pepsin reagent
• Tris-HCL buffer pH- 10
53. Immunohistochemistry in practice
• The choice of technique to suit the needs of particular types of work is
governed by some important factors.
Frozen sections
• Although the use of frozen sections for diagnostic purposes is decreasing,
immunohistochemistry on frozen sections remains an important
histological tool.
Advantage: preserves enzyme and antigen function
Disadvantages:
• Poor morphology
• Limited prospective studies
• Storage of material difficult
• Cutting difficulty over paraffin sections
54. • Poor morphology → improved by ensuring the frozen sections are thoroughly
dried both before and after the sections are fixed in acetone.
• Acetone assists preservation of the antigen and related morphology and
also destroys most harmful infective agents.
55. Cytological preparations
• Acetone-fixed smears are often preferred by the immunohistochemist as
it allows a wide range of primary antibodies to be employed without
destroying the target epitopes
• In alcohol, consequently the number of antigens demonstrable may be
limited, although perhaps the morphology is superior.
56. Blocking endogenous enzymes
• If enzymes similar to those used as the antibody label are present in the
tissue, they may react with the substrate used to localize the tracer and
give rise to problems in interpretation.
• Inhibiting endogenous enzyme activity prior to staining can eliminate
false-positive reactions.
• Tissues incubated with DAB substrate prior to primary antibody
incubation- if tissue turns brown- peroxidase present and blocking steps
needed.
• Incubation in absolute methanol containing 0.5% hydrogen
peroxide for 10 minutes at room temperature.
57. • Most endogenous alkaline phosphatase activity can be blocked by:
Adding levamisole in the final incubating medium.
Using 20% acetic acid can block intestinal alkaline phosphatase.
• Proteins blocked by: 10% normal serum
58. Blocking background staining
• The major causes of background staining in immunohistochemistry are
hydrophobic and ionic interactions and
endogenous enzyme activity.
• Non-specific uptake of antigen, particularly the high affinity of collagen and
reticulin for immunoglobulins, can cause high levels of background staining.
59. Hydrophobic interactions :
• Tissues that give background staining as a result of hydrophobic
interactions include collagen and other connective tissues, epithelium, and
adipocytes.
Prevention:
• Addition of a blocking protein,
• Addition of a detergent such asTriton X ,
• Addition of a high salt concentration, 2.5% NaCl, to the buffer.
• Addiion of the blocking serum to the diluted primary antibody.
60. • Non-specific staining is most commonly produced because the primary
antibody is attracted nonimmunologically to highly charged groups
present on connective tissue elements.
• Prevention: Add an innocuous protein solution to the section before
applying the primary antibody.
• Traditionally, non-immune serum from the animal species in which the
second (bridging) antibody was raised is used as a blocking serum.
61. Controls:
• Controls validate immunohistochemical results.
• It is essential that any method using immunohistochemistry principles
include controls to test for the specificity of the antibodies involved.
• Negative control. This involves either the omission of the primary antibody
from the staining schedule or the replacement of the specific primary
antibody by an immunoglobulin which is directed against an unrelated
antigen.
• Positive control. 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
62. COUNTERSTAINING
The final step in the process is counterstaining and mounting slides.
counterstains used are:Haematoxylin
Hoechst stain and
DAPI (4',6-diamidino-2-phenylindole)
Hematoxylin is used as the nuclear counterstain for most routine
IHC staining.
63. • Hoechst stains are part of a family of blue fluorescent dyes used
to stain DNA. There are three related Hoechst stains: Hoechst 33258,
Hoechst 33342, and Hoechst 34580.
• DAPI (4',6-diamidino-2-phenylindole) is a fluorescent stain. It is used
extensively in fluorescence microscopy. As DAPI can pass through an
intact cell membrane, it can be used to stain both live and fixed cells,
64. Limitations of Immunohistochemistry:
1. Experience: Experience is critical in standardizing the procedure
including the selection and proper dilutions of necessary reagents and
regular performance of all the appropriate controls. Interpretation too has
its foundation in experience.
2. Availability of antibodies: The paucity of antibody with high degree of
specificity for cellular and tissue antigens was serious limitation until
recently. This has been remedied in part by using hybridoma technique for
monoclonal antibodies.
3. Antigen loss: The specificity of an antibody for particular antigen and its
ability to react with that antigen require the preservation of antigen
configuration
65. Current applications of immunohistochemistry
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