Comparison of various immunohistochemical methods. Demonstration of extracellular antigens in cryostat sections.

Immunofluorescence (IF) and immunoperoxidase (IP) methods for the demonstration of IgG bound to intercellular substance in pemphigus skin were compared. Direct and indirect IF and IP methods had comparable sensitivities. The use of undiluted primary antiserum gave false negative results with the indirect methods. This was probably due to the prozone effect. The higher sensitivity of multiple layer techniques permitted the use of lower titers of the primary antiserum and gave positive staining results even in tissue fixed under less than optimal conditions. Enzyme bridge (EB) and peroxidase-antiperoxidase (PAP) methods gave more dermal background staining than the avidin-biotin-peroxidase complex (ABC) method. In addition, the staining results obtained with the EB and PAP methods were dependent on optimum dilution of different antiserum layers against each other. False negative staining was seen as a result of the secondary antiserum being too dilute in relation to the primary antiserum. This was probably because both Fab parts of the link antibody were bound to the primary antiserum. The ABC method was less sensitive in this respect. With all methods, fixation for 5 min at +4 degrees C in acetone gave the best preservation of immunoreactive determinants of IgG. The results indicate that when an immunohistochemical method is chosen for the demonstration of extracellular antigens in cryostat sections, the sensitivity of the method and preservation of the immunoreactive determinants in the sections, the time needed for staining, access to a fluorescent microscope and reproducibility should be taken into consideration.