Simultaneous demonstration of lectin-binding sites and antigens detected by monoclonal antibodies in a parallelized double-staining technique: a highly discriminative and quickly developing technique for frozen sections.

Abstract

A sensitive immunoenzymatic double-staining technique is presented for the simultaneous visualization of lectin-binding sites and antigenic structures detected by monoclonal antibodies. The lectin is demonstrated by an extended unlabeled peroxidase-antiperoxidase (PAP) technique and the monoclonal antibody by an alkaline phosphatase-antialkaline phosphatase (APAAP) method, which corresponds to the standard PAP technique. 3-amino-9-ethylcarbazole (AEC) and fast blue BB salt serve as substrates for the peroxidase and the alkaline phosphatase, respectively. The antisera and the enzyme complexes raised in different animals enable the performance of three parallel incubation steps. The staining procedure requires three and a half hours altogether. This method proved to be highly discriminative and rather insensitive to interference by various artifacts.