Double immunocytochemical staining in the study of antibody-producing cells in vivo. Simultaneous detection of anti-hapten and anti-carrier antibody-producing cells in lymphoid tissue.

Mice were injected intravenously with 2 mg of a bovine gamma globulin-penicilloyl (BGG-Pen) conjugate. Cells producing specific antibodies against the protein carrier bovine gamma globulin (BGG) and cells producing specific antibodies against the hapten penicilloyl (Pen) could be distinguished simultaneously in the same spleen section using a combined peroxidase (HRP) and alkaline phosphatase (AP) immunocytochemical technique. AP-BGG conjugate was used for detection of anti-BGG-producing cells and HRP-human serum albumin (HSA)-Pen conjugate, prepared by coupling penicillin to HRP-HSA conjugate, was used for detection of anti-Pen-producing cells in the same spleen section. After performing both HRP and AP cytochemistry, cells with a blue-stained cytoplasm represent anti-BGG-producing cells and cells with a red-stained cytoplasm represent anti-Pen-producing cells.