Staining Procedures for Ultrathin Sections of Tissues Embedded in Polyester Resin

Abstract

Tissues were fixed for 30 min. in cold (0-2[degree]C) 1% OsO4 (Palade) buffered at pH 7.7, to which 0.1% MgCl2 was added. Dehydration was in a graded ethanol series (containing 0.5% MgCl2) at 0-2[degree]C, and terminated with 2 changes of absolute ethanol. Tissues were then transferred by a graded series to anhydrous acetone. Infiltration of the tissue with Vestopal-W (a polyester resin), is gradual with the aid of graded solutions of Vestopal-W in acetone. The infiltrated tissue is encapsulated and initial polymerization is done under ultraviolet light at room temperature for 8-16 hr. This is followed by final hardening at 60[degree]C for 36-48 hr. Sections (0.2-1/[mu]) were cut, dried on slides, placed in acetone for 1 min. and then treated by either of the following staining procedures: (1) Thionin-azure-fuchsin staining: Flood the preparation with 0.2% aqueous thionin and heat to 60-80[degree]C for 3 min; if the preparation begins to dry, add stain. Rinse in distilled water. Flood the slide with 0.2% azure B in phosphate buffer at pH 9. Heat to 60-80[degree]C for 3 min; do not permit the preparation to dry. Rinse in distilled water. Dip the slide in MacCallum''s variant of Goodpasture''s carbolfuchsin stain for 1-2 sec. Rinse in distilled water. Check the preparation microscopically for intensity of the fuchsin stain. Repeat dips as may be needed to obtain the desired intensity. Rinse in distilled water. Dehydrate quickly in 95% and absolute alcohol; clear in 2 changes of xylene and cover in Permount or similar synthetic resin. (2) Thionin-azure counterstain for the periodic acid-Schiff reaction: Oxidize the tissue in 0.5% periodic acid for 15 min. and transfer to Schiff''s leuco-fuchsin solution for 30 min. Counterstain with 0.5% aqueous thionin for 3 min; wash in distilled water; stain in 0.2% azure B in phosphate buffer at pH 5.5; wash in distilled water; dehydrate; clear and cover as in the first method. For temporary preparations let dry after absolute alcohol and apply a drop of immersion oil directly on the section.