Immunohistochemistry 2 Introduction 3 • 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. Immunohistochemistry 4 Immunohistochemistry Protocol 5 Fixation Peroxide Block Power Block Microscope Embedding Antigen Retrieval Antibody Mounting Microtome Rehydration Super enhancer Dehydration & clearing Baking De-Waxing Polymer HRP Counter Stain 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 Immunohistochemistry Steps – Slide preparation paraffin wax coated slide The IHC India. NM-123 Colon carcinoma 20033/2007 BioGenex • 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 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 The IHC India. NM-123 Colon carcinoma 20033/2007 BioGenex 8 • Choice of Ag retrieval depends on the Ag to be demonstrated. • Heat Induced Epitope Retrieval (HIER) is widely used. Immunohistochemistry Pre-treatment: HIER • Tissues sections are heated to app 1000C • Achieved by • Microwave oven • Pressure cooker • Vegetable steamers • Water bath • Automated Immunostainers 9 • 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 Immunohistochemistry Pre-treatment: Blocking • Peroxide Block • Blocks endogenous peroxidases • 3% H2O2 • The IHC India. NM-123 Colon carcinoma 20033/2007 BioGenex 10 Protein Block • Blocks all non specific sites • Reduces background • 10% Normal serum is used Immunohistochemistry Primary Antibodies Two types of Abs • Polyclonal Abs: The IHC India. NM-123 Colon carcinoma 20033/2007 BioGenex • Produced by injecting an animal with antigen and harvesting the sera • Monoclonal Abs : • Produced by Hybridomas 11 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 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 Immunohistochemistry Detection Methods A B • 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 The IHC India. NM-123 Colon carcinoma 20033/2007 BioGenex • Alkaline Phosphatase • Detection systems: • Direct or Single step Method • Indirect or Two step Method 14 Immunohistochemistry Enzyme Labels The IHC India. NM-123 Colon carcinoma 20033/2007 BioGenex 15 • 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. Immunohistochemistry Counter Staining The IHC India. NM-123 Colon carcinoma 20033/2007 SPECIMEN 16 • 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. Immunohistochemistry Staining result 17 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 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 Immunohistochemistry Troubleshooting • Weak or No staining • Over staining • High Background 20 Immunohistochemistry Troubleshooting: Weak or No staining Sources 21 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 Immunohistochemistry Troubleshooting: Weak or No staining Sources 22 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 Immunohistochemistry Troubleshooting: Over staining Sources 23 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 Immunohistochemistry Troubleshooting: High Background Sources 24 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 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 Thank You Please visit www.biogenex.com for more details on our product portfolio 26
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