Immunohistochemical conditions for staining choline acetyltransferase-containing axon terminals in the rat

Immunohistochemical conditions for staining cholinergic axon terminals using a commercially available anticholine acetyltransferase (anti-ChAT) monoclonal antibody were determined in the rat. A number of variations of procedures including fixative composition, fixation time, and incubation time and temperature in the anti-ChAT antibody solution were tested. Optimal procedures for minimizing the chance of negative staining of ChAT-containing axon terminals consisted of perfusion with a mixture of 4% paraformaldehyde and 0.2% glutaraldehyde for 3 min followed by a fixative containing only 4% paraformaldehyde for 10 min, and reaction with the anti-ChAT antibody for 2 days at 37 C. The distribution patterns of axon terminals stained in the cerebral cortex and the hippocampal formation were comparable to those reported by other investigators using different monoclonal antibodies.