Hot, Acidified Rhodamine B and Methylene Blue; A Differential Stain Dichromatic for Hair Follicular Keratins, Achromatic for Epidermal Keratin

Abstract

Procedure:Cut paraffin sections and float on a 45–50 C water bath; spread silicone-rubber adhesive (Clear Seal-General Electric) thinly and evenly over 2/3 of the slide; pick up the sections from the floatation water with the coated slide; dry for 1.5 hr at 25 C and at 60 C for 0.5 hr; deparaffinize, and hydrate to water. Place 150 mg of rhodamine B and 150 mg of methylene blue each in separate 100 ml beakers and add 80 ml of 10% HCl to each beaker. Bring both solutions to a boil on a hot plate in a fume hood; immerse tissue sections in the boiling rhodamine B exactly 2 min; rinse in a beaker of 10% HCl 5 sec; immerse in the boiling methylene blue exactly 0.5 min; rinse in distilled water; blot dry; and mount in a silicone-rubber medium (Glass and Ceramic Adhesive—Dow Corning Corp.). Hair shaft keratin stains red; inner root sheath keratin and keratogenous zone of the hair shaft, blue green; epidermal keratin remains unstained. Pilomatrixornas show foci of both red and blue green keratin; epidermal and hair sheath (“sebaceous”) cysts remain unstained.