A Rapid Phloxine-Methylene Blue Oversight Stain for Formalin-Fixed Material

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Abstract
A rapid method is described, which yields brilliant and selective differential staining with phloxine-methylene blue. Aqueous phloxine (1%) is treated with hydrochloric acid (1 ml/l gm of phloxine) and the water-insoluble precipitate is washed, dried, and dissolved in acidified 95% ethyl alcohol. Slides are stained for several minutes in this solution (0.2%) followed by brief staining in the usual azure-methylene blue solution and differentiated with colophonium-alcohol. The method eliminates the necessity for prolonged staining at elevated temperatures, reducing the total staining procedure to approximately 15 min. In addition, controlled differentiation with colophonium-alcohol can be carried out on formalin-fixed material without loss of phloxine. The selectivity and tinctorial attributes of phloxine are also considerably improved.

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