Double immunocytochemical staining in the study of antibody-producing cells in vivo. Detection of specific antibody-producing cells in the spleen and simultaneous determination whether or not they produce immunoglobulin G antibodies.

Rabbits were primed intravenously with human serum albumin (HSA) and boosted with the same antigen 2 months later. Cells producing specific antibodies against HSA could be detected in vivo and it could be determined whether or not they belonged to the immunoglobulin (Ig) G class using a combined peroxidase (HRP) and alkaline phosphatase (AP) immunocytochemical technique. HRP-HSA conjugate was used for detection of anti-HSA-producing cells and AP-sheep anti-rabbit IgG (SRIgG) was used to determine the IgG class of the antibodies produced by these cells in the same spleen section. After performing both HRP and AP cytochemistry, cells with a red-stained cytoplasm represent anti-HSA-producing cells not stained for their antibody class and cells with a blue-stained cytoplasm represent cells producing IgG antibodies not directed against HSA. Cells with a double-stained cytoplasm represent cells producing anti-HSA antibodies belonging to the IgG class. We also attempted to determine whether or not part of the anti-HSA-producing cells belonged to the IgM class using AP-sheep anti-rabbit IgM (SRIgM). In this case no double-stained cells were detected, indicating that the affinity of intracellular IgM-anti-HSA antibodies is too low to allow detection using the present technique.