A simple two-step immunocytochemical method using protein A-peroxidase to stain immunoreactive cell antigens

Abstract

We report a simple two-step immunocytochemical method that uses staphylococcal protein A conjugated to horseradish peroxidase and the H2O2-3,3'-diaminobenzidine chromogenic reaction to stain neurons and fibers containing immunoreactive cell antigens. Our protein A-peroxidase method was found to produce stains of equal or superior quality to those of the classical 3-step peroxidase-anti-peroxidase (PAP) technique. Since the chromogenic reaction with protein A-peroxidase has low non-specific tissue binding, the reaction can be carried out at acidic pH with higher horseradish peroxidase activity and no significant increase in background. Using this method, we were able to produce clear stains of neurons and fibers containing tyrosine hydroxylase or fibers containing methionine-enkephalin. The use of protein A-peroxidase conjugate substantially simplified the immunocytochemistry procedure, and reduced both the time and cost of experiments.