Basics of Immunohistochemistry (IHC)
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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.
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Basics of Immunohistochemistry (IHC)
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- 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
- 5. Immunohistochemistry Protocol 5 De-WaxingDe-Waxing RehydrationRehydration Antigen Retrieval Antigen Retrieval Peroxide Block Peroxide Block Power BlockPower Block Counter Stain Counter Stain Dehydration & clearing Dehydration & clearing MountingMounting MicroscopeMicroscopeFixationFixation EmbeddingEmbedding MicrotomeMicrotome BakingBaking Antibody Super enhancer Polymer HRP
- 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 6
- 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 9
- 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 10 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 19
- 20. Immunohistochemistry Troubleshooting • Weak or No staining • Over staining • High Background 20
- 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 25
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