Prestaining Oxidation by Acidified H2O2 for Revealing Schiff-Positive Sites in Epon-Embedded Sections

Reliable production and identification of Schiff-positive sites on glutaraldehyde-osmium fixed 0.5-1 μsm Epon sections is accomplished by preoxidation of sections with 10% H₂O₂ acidified with H₂SO₄ (HPSA) to pH 3.2 (Pool, C. R., Stain Techn., 44: 75-9, 1969). Light green as a counterstain is used. Steps in the procedure are: HPSA, 1-2 min at 25-30 C; washing; 1% light green 3-5 min; brief rinse; Schiff reagent 1-3 min; washing; drying; clearing in xylene and mounting in resin. The use of acidified H₂O₂ prevents the common occurrence of Schiff background staining in glutaraldehyde-fixed tissues and permits optimum penetration of staining solutions. Sections were attached to glass slides without adhesive and were processed in Coplin jars. Prior to drying, excess solutions should be drained and wiped away with lens tissue to prevent formation of precipitate on the sections.