Increasing membrane contrast by means of imidazole‐osmium post‐fixation as exemplified by skeletal muscle fiber

Abstract

Until now, the interpretation of findings derived from investigations on membrane structures (T tubules, sarcoplasmic reticulum, the Golgi apparatus) in thick sections of mammalian muscle tissue has been limited in TEM due to the lack of sharp resolution of the membrane contours. This article shows how the imidazol-osmium post-fixation of tissue blocks can be used to achieve well-contrasted, sharply defined membrane contours. Therefore, unstained sections from imidazol-osmium post-fixed tissue can be examined immediately. But protein structures (e.g., ribosomes) remain uncontrasted with this technique. If needed, it is possible to visualize the protein structures by conventional section staining with uranyl acetate and lead citrate. This method is suitable for both ultrathin and thick sections (>150 nm).

The Transillumination Possibility of Imidazole–Osmium Postfixed Tissue and Its Consequences for the Handling of Tissue Samples

“…The advantages of the imidazole-osmium postfixation for investigations in the TEM are described elsewhere~Angermüller & Fahimi, 1982;Voigt et al, 2002Voigt et al, , 2003 Voigt et al, 2002!. Also in the jejunum, the cell membranes as well as lipid droplets and chylomicrons are stained more strongly in the imidazole-osmium postfixed samples, so that investigations can be done without further staining of ultrathin sections.…”

About the morphological relationships of the sarcoplasmic reticulum in the sole plate area of the frog

“…As in mammals (Voigt et al, 2002) the use of the imidazole-osmium postfixation proves to be suitable for a sharp membrane outlining at frogs. Furthermore, the blackening of the sarcoplasm of sole plate, mainly caused by the glycogen, is suppressed.…”

Perisynaptic Schwann cells of the vertebrate motor endplate bear modified cilia

“…The investigations were carried out using the diaphragm dissected from mice and the sartorius muscle dissected from frogs (Dauber and Meister, 1986;Dauber et al, 1999Dauber et al, , 2000Voigt et al, 2002). Unless otherwise mentioned, a 0.1 mol cacodylate buffer was employed as buffer solution.…”

“…Anesthetized mice (n ϭ 7; NMRI ϩ wildtype) were perfused and fixed with 2.5% glutaraldehyde from the left ventricle (for further details, see Dauber and Meister, 1986). The middle third of the diaphragm, including the MEPs (Bowden and Duchen, 1976), were divided into portions and postfixed at 4°C by one of the following procedures: 1) our routine procedure using 1% osmium tetroxide for 2 hours (Dauber and Meister, 1986); 2) the imidazoleosmium procedure using 1% osmium tetroxide in 0.1 mol imidazole for 1 hour (Voigt et al, 2002); and (3) lanthanum incubation using 1% osmium tetroxide with 0.08% potassium ferrocyanide for 3 hours and incubation thereafter for 1.5 hours in 1% lanthanum nitrate .…”