The resolution of the electron microscope was so high that the cytoplasmic components were readily identified in the cytoplasm. Osmium tetroxide vapor staining gave better contrast in the images of the specimens.
Summary : A mixture of formaldehyde and glutaraldehyde as a fixative in conjunction with GMA-Quetol 523 embedding readily permits the preservation of semi-thin sections for routine use in combined light and electron microscopy. Combined 4% formaldehyde-2% glutaraldehyde fixation gives excellent preservation, and general and histochemical staining performed at the light microscopic level provides satisfactory and GMA-Quetol 523 sections in this range, nuclear and cytoplasmic components such as the nucleoli, mitochondria, Golgi apparatus and cytoplasmic granules, were clarified with various basic and acid dyes, especially hematoxylin and eosin. DNA, glycogen and other polysaccharides were also sufficiently well preserved to studyThe effectiveness of glutaraldehyde, formaldehyde, formaldehyde-glutaraldehyde, periodate-lysine-paraformaldehyde, Bouin's and Zenker's solution on GMA-Quetol 523 embedded tissues was compared. Formaldehyde-glutaraldehyde mixtures promise to be a very useful fixative for use in GMA-Quetol 523 embedded and are suitable for the semi-thin sections necessary in combined light and electron microscopic studies. The results were both visually pleasing and ideal for photomicrography.
Semithin sections embedded in water-miscible methacrylates were used for the study of fine structures of cells and tissues in the central nervous system by light microscopy instead of the conventional paraffin sections. This method used a water-miscible methacrylate mixture consisting of 2-hydroxypropyl methacrylate (HPMA), Quetol 523 and methyl methacrylate (MMA) as an embedding medium. The mixture had a low viscosity, was easy to handle and penetrated readily and completely into the specimen, producing a homogenous block from which it was easy to make sections 1.5 microns thick. Staining could be localized far more precisely in these sections than in paraffin sections owing to the thickness of the semithin sections and to the excellent structural preservation of cellular components.
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.
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