Stain Technology: A Phenylhydrazine-Leucofuchsin Sequence for Staining Nerves and Nerve Endings in the Integument of Poultry

Abstract

This sequence for staining cutaneous nerves and nerve endings uses 1% formic acid as a fixative for 1 hr, followed by two treatments of 5 min each in 6% H2SO4. The tissue is then submerged in fresh 5% phenylhydrazine hydrochloride for 30 min, washed in running tap water for 10 min, and given a 5 min soak in distilled water. The specimen is placed in Lillie's “cold Schiff” reagent for 4 hr; transferred to 6% H2SO4, 4 changes of 5 min each; washed in distilled water, 3 changes of 5 min each; dehydrated in acetone, 4 changes of 10 min each; and cleared in 2 changes of methyl benzoate, the 1st for 1 hr and the 2nd until the tissue clears. Nerve fibers stain pinkish-purple; muscles also take up the stain, yet the nerves are discernible from the muscles. All other tissue remain unstained.