An osmium-free method of epon embedment that preserves both ultrastructure and antigenicity for post-embedding immunocytochemistry.

Abstract

Immunocytochemistry for amino acids with post-embedding gold is compatible with glutaraldehyde fixation, osmication, and embedding in epoxy-based plastics, but immunogold detection of larger molecules in the central nervous system commonly requires special procedures, e.g. minimizing exposure to glutaraldehyde, eliminating osmium, cryosectioning, and/or embedding in acrylic plastics. These make samples more difficult to prepare and view and may compromise structural preservation. We report a new technique, fixing with high levels of glutaraldehyde, replacing osmium with tannic acid followed by other heavy metals and p-phenylenediamine, and embedding in Epon. This method optimizes antigenicity while retaining the structural preservation and convenient handling of standard embedding techniques. Compared to standard Epon embedment, labeling for neuropeptides in brain and spinal cord is improved. Moreover, the present method yields excellent labeling of glutamate receptors (difficult to identify with traditional post-embedding techniques) and enables simultaneous visualization of associated neurotransmitters.