Color-contrast staining of two different lymphocyte subpopulations: a two-color modification of alkaline phosphatase monoclonal anti-alkaline phosphatase complex technique.

A dual staining method for different human lymphocyte subpopulations with nonoverlapping antigen distribution patterns is described. Cytocentrifuge slide preparations of peripheral blood nonadherant mononuclear cells (NAMNC), bone marrow aspirate or buffy coat smears were fixed in acetone and incubated with a primary mouse monoclonal antibody (MAb) against a lymphocyte antigen (CD8, Ig-light-chain, CD19, CD4) followed by rabbit anti-mouse immunoglobulin (Ig) and the alkaline phosphatase monoclonal anti-alkaline phosphatase (APAAP) complex. After repeating the "bridge" antibody and the APAAP, a red product was developed with fast red TR-naphthol AS-BI phosphate. Following this one-color stain the process was repeated using a different primary mouse MAb against another lymphocyte antigen (CD4, Ig-light chain, CD3, MHCII DR, CD5) and fast blue BB-naphthol AS-MX phosphate at the last step to yield a blue product. Control slides stained by the standard one-color APAAP method with the relevant primary MAb showed that there was no nonspecific labelling and the percent of positive cells in a given test was almost identical. To achieve an intense blue in the second stain for some antigens, e.g., CD4, either the MAb concentration had to be increased or two different MAbs recognizing differing epitopes of the same antigen, e.g., T1 and UCHT2 for CD5, were applied. Any change of red to purple at the site of the first stain after 15 min exposure to the blue-yielding AP substrate is due to residual AP activity of the first stain rather than to crossbinding of immunoreagents.(ABSTRACT TRUNCATED AT 250 WORDS)