A comparison of ABC, unlabeled antibody and conjugated immunohistochemical methods with monoclonal and polyclonal antibodies--an examination of germinal center of tonsils.

Staining of lymphoid tissues with anti-immunoglobulin antibodies often is complicated by heavy interstitial staining and potential unwanted binding through Fc receptors. This problem is aggravated in the germinal center because of an unusually intense extracellular staining. In an attempt to examine a variety of staining methods, we found both poly- or monoclonal antibodies produced satisfactory results only if a suitable method was employed, such as a two-step or three-step avidinbiotin-peroxidase complex (ABC) method. Other staining technics for example, indirect conjugate or four-layer PAP methods, failed to provide a clean background. The use of F(ab')2 fragments did not significantly improve the staining quality, indicating that the role of the Fc receptors in inducing nonspecific staining is not significant in acetone-fixed frozen tissues. Germinal centers also were studied with a panel of monoclonal and polyclonal anti-Ig antibodies, and the results indicated that the number of surface or cytoplasmic Ig-positive cells in the germinal center is variable from one follicle to another. IgG+ cells are generally sparse (less than 10%) and are scattered in the light zone of follicles. IgA+ cells constitute about 0-10% of the total cell population and tend to be distributed randomly either in the dark or light zones. IgM+ and IgD+ cells may be scanty (less than 10%) or abundant (50-70%). The IgM+ and IgD+ cells tend to localize in the dark zone of the follicles. The results indicate that, depending on the stage of differentiation or transformation of the germinal center, s-Ig or c-Ig expression is highly variable.