Week 4 H&C Immuno Histochemistry PDF

Basic Concepts in Immunohistochemistry Histology and Cytology Course AY2022-2023 Semester 1 – for MLS 2020 group Oman College of Health Sciences  An introduction to IHC Immunohistochemistry (IHC) is the localization of antigens in tissue sections by the us...

Basic Concepts in Immunohistochemistry Histology and Cytology Course AY2022-2023 Semester 1 – for MLS 2020 group Oman College of Health Sciences  An introduction to IHC Immunohistochemistry (IHC) is the localization of antigens in tissue sections by the use of specific labeled antibodies. These antigen-antibody interactions are then visualized by markers such as a fluorescent dye, an enzyme, a radioactive element or by colloidal gold. IHC was first used in 1941 by Albert H Coons and his colleagues. 2  Basic IHC Principle Cellular Added Immune antigen antibody complex Immune Enzyme or Soluble complex Fluorescent Coloured dye substance Soluble Chromogen Insoluble Coloured (ex: Diamino- Coloured substance Benzidine) substance 3  IHC Basic Protocol Tissue Fixation & Processing 3-5 micron thick section on coated slides Deparaffinization (Dewaxing) & Rehydration Antigen Retrieval – HIER or PIER Blocking of Endogenous Peroxidase Application of Primary Antibody Application of Secondary Antibody Application of substrate & chromogen depending on method used Application of chromogen – DAB / AEC / NBT Counterstain with Haematoxylin / Nuclear Fast Red / Methyl Green Dehydration, Clearing, and Mounting 4  Visualization / substrate techniques Immuno Enzyme Immuno Histochemistry Fluorescence  The substance attached Histochemistry to the antibody is an  The substance ENZYME attached to the  The enzyme commonly antibody is a used is Horse Radish FLUORESCENT DYE Peroxidase (HRP) or  Fluorophores used Alkalnine Phosphatase include FITC, Texas  Result can be seen with Red, Rhodamine or Light microscope DyLight Fluor  Most commonly used  Result can be seen method with Fluorescent microscope 5  Tissue Fixation 10% NBF( FFPE tissue) 4% paraformaldehyde in 0.1M phosphate buffer 2% paraformaldehyde with 0.2% picric acid in 0.1M phosphate buffer PLP fixative: 4% paraformaldehyde, 1.2% lysine, and 0.2% per-iodate in 0.1M phosphate buffer 4% paraformaldehyde with 0.05% glutaraldehyde (TEM immunohistochemistry) 6  IHC Enzyme Methods Direct Method Indirect Method PAP (Peroxidase Anti-Peroxidase) Method ABC (Avidin-Biotin Complex) Method LSAB (Labeled StreptAvidin Biotin) Method Polymeric Methods EnVision System ImmPRESS System CSA (Catalyzed Signal Amplification) Methods CSA I (utilizes biotinyl-tyramide) CSA II (utilizes fluorescyl-tyramide) 7  Antigen Retrieval To unmask hidden antigenic sites To break protein cross-links formed by formalin fixation Techniques: Heat Induced Epitope Retrieval (HIER) Application of heat to formalin-fixed, paraffin wax-embedded tissue using microwave ovens, pressure cookers, steamers, etc Proteolytic Induced Epitope Retrieval (PIER) Application of enzyme digestion processes using enzymes such as proteinase k, trypsin, chymotrypsin, pepsin, pronase, etc 8  Blocking of endogenous peroxidase Some cells or tissues contain endogenous peroxidase. Using of HRP conjugated antibody may result in high, non-specific background staining. This non-specific background can be significantly reduced by pre-treatment of cells / tissues with 0.3 % – 3.0% hydrogen peroxide prior to incubation with HRP conjugated antibody. 9  Application of Primary and Secondary Antibodies Depends on method used: Direct Method Indirect Method PAP (peroxidase anti-peroxidase) Method ABC (Avidin-Biotin Complex) Method LSAB (Labeled StreptAvidin Biotin) Method Polymeric Methods CSA (Catalyzed Signal Amplification) Methods 10 Direct Indirect PAP Method Method Method 11 ABC (Avidin-Biotin Complex) Method 12 Polymer-based Method 13  Uses of IHC In identifying cancer cells In differentiating Carcinomas from Sarcomas In typing Leukaemias In identifying secondary tumour deposits In determining receptor status (ER and PR) in breast carcinomas In localizing specific protein expression in neurobiology 14 Diagnostic IHC Markers – a few examples Cytokeratin – for carcinomas LCA – for leukaemias CD15 & CD30 – for Hodgkin’s disease AFP – for hepatocellular carcinoma CD117 – for Gastrointestinal stromal tumours PSA – for prostate cancer Desmin – for non-epithelial tumours CD20 – for B-cell lymphomas CD3 – for T-cell lymphomas GFAP – for brain tumours Myosin – for muscle tumours S100 Protein – for melanomas 15 16 17 18 19 20 21