PRINCIPLE AND PURPOSE OF IMMUNOSTAINING:
Immunohistochemistry (IHC) is in situ-detection
of antigens in tissue sections and cells by lectins, monospecific monoclonal
and polyclonal antibodies (Abs). This concept has been extended
to in situ hybridization with haptenated probes. Detection is obtained
by visualization of the antigen by a light microscopy-detectable opaque
dye of pathologist's choice.
Immunofluorescence (IF) is the same technique
but the antigen is visualized with a fluorochrome-tagged molecule or chain
of molecules; the fluorochrome is then excited and viewed with a special
light source and a system of selective filters.
This technique has been for long time pathologist's unfriendly because of lack of morphologic detail and need of a specialized microscope and dark environment. This has changed recently Ref, particularly in experimental pathology.
Most of the specimens stained in a routine immunohistochemistry
lab in Surgical Pathology are formalin-fixed, paraffin embedded tissues.
Fixation and embedding cause antigen masking, but also better retention
of labile proteins, nucleic acids and small peptides. In order to overcome
the drawback of antigen loss, enzymatic- or heat- mediated antigen retrieval
is used (Ref. 1,
2,
3,
4
) (see a comprehensive ref. list here).
This lab is committed to apply and to improve these techniques, in an effort
to reach the highest sensitivity and specificity in routine diagnostic
immunostaining.