Hematoxylin-Eosin Staining of OsO4-Fixed Epon- Embedded Tissue; Prestaining Oxidation by Acidified H2O2

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Abstract
Successful application of hematoxylin-eosin staining to 0.5-1 μ sections of OsO4-fixed Epon-embedded mammalian tissue is made possible by first treating the sections for approximately 1 min at 25-30 C with 10% H2O2 acidified with 0.1 or 0.01 N H2SO4 to pH 3.2. Subsequent steps are: washing; drying; Hams hematoxylin at 50 C, 1-2 min; washing; drying; 0.2–0.3% NH4OH in 70% ethanol, 3-5 sec, drying at 50 C; 5% aqueous eosin for 3 & 45 sec at 25-30 C, washing; drying; clearing in xylene and mounting in resin. The use of acidified H2O2 prevents the staining of Epon and permits the characteristic staining picture to be obtained. Sections were attached to glass slides without adhesive and processed horizontally on a rack. Slides should be well drained and blotted before each drying step, to prevent formation of precipitate on the section.