Combination of the peroxidase anti-peroxidase (PAP)-and avidin-biotin-peroxidase complex (ABC)-techniques: an amplification alternative in immunocytochemical staining

Summary

A combination of the PAP- and ABC-techniques was developed to enhance the intensity of the immunocytochemical staining with monoclonal antibodies at light and electron microscopical levels. This amplification technique could be performed in 4 (single PAP + ABC) or 6 (double PAP + ABC) sequential steps depending on the quality of the primary antibodies used and the processing of the tissue before the immunocytochemical reaction:

First step — Incubation of the tissue sections with the monoclonal primary antibodies; Second step — biotinylated anti-rat or anti-mouse IgG; Third step — monoclonal PAP complex; Fourth step — ABC complex which binds to the biotinylated secondary antibody. If stronger enhancement of the immunostaining has required the steps 2 and 3 could be repeated followed by the 6th step — the ABC complex.

Choline acetyltransferase-like immunoreactivity of the rat hypoglossal nucleus and desmin- and vimentin-like immunoreactivity of human testis were studied. After the 4-and more pronounced the 6-step reaction a significant increase of the staining intensity was observed for all the reactions under study. ChAT-like immunoreactivity was observed to longer distances of the nerve cell dendrites after their emerging from the perikarya and within a greater number of structures in the neuropil as compared to the standard techniques. At electron microscopical level the technique permits longer fixation of the tissue which is important for the better preservation of the ultrastructure as well as for the easier recognition of the reaction product even in the smallest dendrite branches and the axons of the nerve cells. Stronger staining intensity was obtained for desmin-like immunoreactivity (LI) (within myofibroblasts of the lamina propria and muscle cells of the blood vessels)-and vimentin-LI (within Sertoli cells, myofibroblasts of the lamina propria and fibroblasts of the interstitium) of the human testis. The full combination (6 step-reaction) permits the detection of smallest quantities of an antigen in sections of different tissues using highly diluted primary antibodies. The technique represents a further alternative among the immunocytochemical amplification methods.