Semithin sections, cut from tissues stained with acid and basic dyes after embedding in 2-hydroxypropyl methacrylate, Quetol 523 and methyl methacrylate, showed cytoplasmic components at a high resolution by light microscopy. These same sections could then be viewed, after osmium tetroxide, uranyl and lead staining, by the electron microscope. These sections had a number of inherent advantages: they could be observed with a light microscope; they facilitated analysis of cellular structures in the identical sites, and they were frequently the optimum thickness to provide three-dimensional information. We clearly established the structural detail of this same-section correlative light-electron microscopy approach by showing that the coloured materials observed in such sections of cells followed the distribution of fine structures within the same sections as determined by electron microscopy. In some instances the fidelity of the correlation between the distribution of the coloured area and cytoplasmic components in identical cells of the same section revealed significant details which could not visualized in thin sections. This technique, therefore, provided a simple and useful solution to many problems that require the localization of cellular components in identical cells selected previously by light microscopy.
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.
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