Microscopy Special Techniques in Microscopy: Immunohistochemistry

The process for detecting antigens (Ags) in histological material – uses antibodies (Abs) directed against specific antigenic sites in cells and tissues – labeling with colouring agents (or fluorescence, or electron opaque material) to visualise Ag-Ab reaction products

History of Immunocytochemistry

Immunohistochemical methods Direct method – antigen + [antibody + marker substance] Indirect method – antigen + 1° antibody + [2° antibody + marker substance] Indirect method is amplification process – large amount of marker substance can be attached Most widely used method – PAP and ABC methods

Fluorescent ICC Direct method – antigen + [antibody + fluorescent dye] Indirect method – antigen + 1° antibody + [2° antibody + fluorescent dye] fluorescent dye is often fluorescein isothiocyanate (FITC)

ICC methodology

Chromagen localisation ABC-complexed chromagen (red) localising estrogen receptors in breast tumor. Tissue is counter stained with haematoxylin to show nuclei.

Fluorescent ICC

Enzyme-labelled antibody methods Immunoperoxidase method – Nakane and Pierce (1966): introduced a method for the application of enzymes as markers of antibodies in cells and tissues – Graham and Karnovsky (1966): developed a method for histochemical demonstration of horse-radish peroxidase enzyme (HRP) at ultrastructural level HRP conjugation technique antigen + [antibody + coupling agent + HRP] – p,p ’ -difluoro-m,m ’ -dinitrophenyl sulphon (FNPS) – glutaraldehyde – sodium periodate

Peroxidase-Antiperoxidase (PAP) Designed to overcome the problems inherent in fluorescent labelling – Signal reduction in fluorescent staining (bleaching) wavelength stimulation time – Must use fluorescent microscope expensive special filters – Cannot be used in electron microscopy

PAP technique - 1 Antigen + 1º antibody + 2º antibody + PAP complex Prestaining preparation – Remove Fixatives wash thoroughly with PBS sodium borohydrate pretreatment – Enhancing penetration of antibodies LM -- use detergent (Triton X-100, etc.) EM -- use graded ethanol – Blocking non-specific antibody determinants pretreatment with animal serum – normal goat serum (NGS) – bovine serum albumin (BSA)

PAP technique - 2 Remove fix Reaction with primary antiserum – Primary antibody (polyclonal or monoclonal) human or animal (X) Ag animal (Y) Ag Y-anti X Ab determination of 1º antibody dilution Reaction with secondary antiserum – Bridging process primary antibody + secondary Ab + PAP complex – Secondary antibody: Z origin anti-Y IgG one Fab directed primary Ab other Fab directed toward Y origin PAP complex

PAP technique - 3 Reaction with PAP complex – Amplification process Y origin PAP complex (peroxidase Y-antiperoxidase complex) anti-Y secondary Ab + Y PAP complex – Visualisation of PAP end products DAB (3,3 ’ -diaminobenzidine) - an insoluble phenazine polymer – oxidative polymerisation - serves as electron donor Peroxidase Hydrogen peroxide (H 2 O 2 ) - substrate

PAP localisation Substance P from dorsal horn of rat spinal chord.

Avidin-biotin-peroxidase complex (ABC) method Alternative method to PAP technique – More sensitive than PAP method amplification is much greater more staining sites – Based on avidin-biotin affinity avidin with Mol Wt = 7000 biotin (vitamin H) – More convenient availability of ABC kits from several companies

ABC technique - 1 Antigen + 1º antibody + biotin-labelled 2º antibody + HRP- labelled streptavidin Prestaining preparation – Remove Fixatives wash thoroughly with PBS sodium borohydrate pretreatment – Enhancing penetration of antibodies LM -- use detergent (Triton X-100, etc.) EM -- use graded ethanol – Blocking non-specific antibody determinants pretreatment with animal serum – normal goat serum (NGS) – bovine serum albumin (BSA)

ABC technique - 2 Reaction with primary antiserum – Primary antibody (polyclonal or monoclonal) human or animal (X) Ag animal (Y) Ag Y-anti X IgG Reaction with biotin-labelled secondary antiserum – Bridging process 1º antibody + biotin-labelled 2º Ab + PAP-streptavidin complex Secondary antibody: Z origin anti-Y IgG Reaction with PAP-streptavidin complex – biotin + streptavidin-PAP (strong biotin reaction) Visualisation of PAP end products – DAB, peroxidase, H 2 O 2

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