The Use of the Benzidine Method on Thick Specimens

Abstract

A method, employing benzidine, nitroferricyanide and H2O2, for demonstrating blood vessels, was applied to formalin-fixed specimens of whole mouse organs and to rat embryos ranging in thickness up to 5 mm. This staining technic outlined the blood vessels by reacting with the Hb of intraluminal erythrocytes. The specimens were treated in a mixture of 5% benzidine in 2.5% acetic acid, and 5% aqueous Na nitroferricyanide; then in dilute H2O2 (4-6 drops H2O2 to 100 ml. water). After dehydrating in alcohol and dioxan, clearing in cedar oil or xylol they were mounted in balsam or stored in cedar oil. The staining procedures were carried out at 37[degree] C.