En bloc staining available for stereoscopic observation of epoxy resin Quetol 651-embedded thick sections under a 300 kV TEM

Abstract

A staining method has been devised for easy en bloc staining for stereoscopic observation of epoxy resin Quetol 651-embedded thick sections under a 300 kV transmission microscope (TEM). In order to enhance staining properties in thick section, osmium tetroxide-fixed tissue blocks are stained only en bloc, since the images of both sides in thick section give high contrast and the image of an intermediate layer shows low contrast by double staining.This method uses carbohydrazide (Polysciences, Inc., U.S.A.) as osmium bridging agent, and both osmium tetroxide and uranyl acetate as electron staining agents.The following procedure is suitable for en bloc staining. 1.Fix small tissue blocks in 2% cacodylate-buffered osmium tetroxide (pH 7.4) for 3 hours at 4°C.2.Wash well in buffer for 1 hour.3.Transfer in 1% aqueous carbohydrazide for 2 hours at room temperature.4.Wash well in distilled water for 1 hour.5.Stain in 1% aqueous osmium tetroxide for 2 hours at room temperature.6.Wash well in distilled water for 1 hour.7.Dehydrate in 50% alcohol for 1 hour.8.Stain in a 2.5% solution of uranyl acetate in 50% alcohol for 3 hours at room temperature.9.Wash in 50% alcohol for 1 hour.10.Dehydrate with 60%, 70%, 80%, 90% and 100% (2 changes) alcohols for 30 minutes each.11.Embed in a mixture of Quetol 651 (Nissin EM Co., Ltd., Japan), nonenyl succinic anhydride, methyl nadic anhydride and DMP-30 according to the method of Kushida et al.