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Microscopy

Microscopy. Special Techniques in Microscopy: Immunohistochemistry. Immunohistochemistry. The process for detecting antigens (Ags) in histological material uses antibodies (Abs) directed against specific antigenic sites in cells and tissues

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Microscopy

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  1. Microscopy Special Techniques in Microscopy: Immunohistochemistry

  2. 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

  3. History of Immunocytochemistry

  4. 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

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

  6. ICC methodology

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

  8. Fluorescent ICC

  9. 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

  10. 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

  11. 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)

  12. 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

  13. 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 (H2O2) - substrate

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

  15. 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

  16. 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)

  17. 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, H2O2

  18. Suppliers • http://www.vectorlabs.com/index.htm

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