Microscopy

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Microscopy

• Special Techniques in Microscopy
• Immunohistochemistry

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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

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History of Immunocytochemistry
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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

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Fluorescent ICC

• Direct method
• antigen antibody fluorescent dye
• Indirect method
• antigen 1 antibody 2 antibody
  fluorescent dye
• fluorescent dye is often fluorescein
  isothiocyanate (FITC)

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

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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

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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)

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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

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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

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

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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)

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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

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