A combined cytochemical and immunocytochemical method for simultaneous visualization of cytoplasmic enzyme reactivity...

Immunocytochemical methods were used in combination with enzyme cytochemistry to visualize simultaneously cytoplasmic enzyme reactivity (for dipeptidyl[amino]peptidase [DAP IV], acid phosphatase [AcP], chloroacetyl esterase [CAE]) and cell surface antigens (Leu-3a, Leu-4, Leu-14, Leu-M1, OKT4, OKT8, OKB7) in cytospin preparations from cell suspensions of human reactive lymphoid tissues (four lymph nodes and three tonsils). Different fixative solutions were tested. Enzyme and immunocytochemical reactions were carried out in different orders of sequence to establish which was the better direction for the combination of the two methods. The following immunocytochemical methods were tested: three stages, avidin-biotin complex, peroxidase-antiperoxidase, alkaline phosphatase-antialkaline phosphatase (APAAP) (using both peroxidase and alkaline phosphatase as labeling enzyme). Acetone or buffered formalin acetone gave the best results both for cytochemical and immunologic reactions. DAP IV and AcP reactivities could be visualized only when cytochemical reactions were performed before immunocytochemistry. CAE reactivity could be demonstrated either before or after immunocytochemistry. Cell surface antigens could be demonstrated with most immunocytochemical methods: however, the APAAP method was preferred for its sensitivity and effectiveness when combined with enzyme cytochemistry. By this approach, cells expressing only immunologic markers and cells expressing only cytochemical markers could easily be distinguished from those coexpressing both markers, because cytochemistry and immunocytochemistry could be combined without affecting the reactivity of each marker, and the reaction products did not hamper the interpretation of preparations.