Immunohistochemistry

Immunohistochemistry is the demonstration of antigens in tissue sections or smears by the use of specific immunological (antibody-antigen) interactions culminating in the attachment of a visible marker to the antigen. The visual marker may be a fluorescent dye, colloidal metal, hapten, radioactive marker or more commonly for light microscopy an enzyme. Ideally, maximal signal strength along with minimal background or non-specific staining are required to give optimal antigen demonstration.

Antigen affinity-purified polyclonal antibodies are recommended for immunohistochemistry because they are 10-fold more potent than the traditional protein G affinity-purified polyclonal antibodies. The use of analyte-specific affinity chromatography greatly reduces or totally eliminates non-specific immunoglobulins. The benefits of using antigen-affinity purified polyclonal antibodies are increased staining intensity and reduced non-specific staining.

At MDS, for each project optimal working dilution of antibodies and all other reagents will be determined. Due to accumulation of autofluorescent pigment lipofuscin in primate and human neuronal tissues, the use of fluorescent probes, for example, FITC or Cy3 is not recommended. Protocols using chromogens ( e.g. , DAB, AEC or immunogold-silver staining) may be used instead.

A selected list of markers for different tissues include:

• Epithelium: Keratins
  • pan-keratin
  • antibodies to keratins of different molecular weights
• Supporting connective tissues:
  • Vimentin--fibroblasts, blood vessels
  • vWF, CD31 (PECAM)-- endothelial cells of blood vessels
• Hematopoeitic tissues
  • CD45
  • B220
  • CD3
  • F480
  • Mac-1
  • Gr-1
  • CD41
• Muscle:
  • Desmin
  • smooth muscle actin
• Neural:
  • GFAP
  • NeuN
  • F480/Mac-1
  • MBP
  • NSE, S100
• Hormones:
  • specific antibodies
  • insulin,
  • casein,
• Germ cells:
  • alpha-feto protein (teratomas)
• Proliferation markers
  • Ki-67