A method of combining biocytin tract-tracing with avidin-biotin-peroxidase complex immunocytochemistry for pre-embedding electron microscopic labeling in neonatal tissue.

A new method that allows the combination of avidin-biotin-peroxidase visualization of antigens and silver-intensified gold labeling of biocytin, a rapid tract-tracer, is described. The method provides a practical tool for in vivo and in vitro studies of chemically specified afferent-target relationships and particularly in developing neural pathways where biocytin is invaluable as a rapidly transporting, sensitive tracer requiring little permeabilizing agents. Transported biocytin was first visualized with silver-intensified colloidal gold conjugated to anti-biotin IgG. This was followed by blocking of all unbound biotin groups of biocytin in the tissue with an Avidin-Biotin blocking kit. Finally, a second antigen, neuronal nitric oxide synthase NOS or GluR2/3 subunit of AMPA receptors, was visualized selectively with avidin-biotin-peroxidase/DAB. This protocol allowed visualization of two chromagens that could be distinguished by electron microscopy. The presence of biocytin was evident by silver particles, while accumulation of peroxidase reaction product marked only the antibody labeling: no cross-reaction between biocytin and the avidin-biotin-peroxidase was observed.