Osmium tetroxide membrane fixative

Figure 10.8 Milk lipid globule membranes released by churning of washed globules and collected by ultracentrifugation retain densely staining coal material along one face of the bilayer membrane. As seen in this electron micrograph of glutaraldehyde and osmium tetroxide-fixed material, the...

The immunoreplica technique (14) is used when it is necessary to detect antigenic sites on the plasma membrane of cultured cells. The cells are cultured on coverslips, and are fixed as described above depending on the antibody in question, and immunolabeled in situ as described in Section 3.1.1.2., steps 3-9. After immunolabeling (Section 3.1.1.2., step 9), they are further fixed with 1% osmium tetroxide and are dehydrated in a graded series of ethanol (70, 90, 100%), critically point-dried, and replicated with a layer of carbon and platinum, The replicas are cleaned with sodium hypochlorite and chronic acid before examination with the transmission electron microscope. Large areas of the replicated plasma membrane remain intact for observation. Colloidal gold probes are probably the only probes of sufficient density that can be detected on these surfaces. 

Electron diffraction patterns have been obtained of myelin and rat central nervous system membranes after osmium tetroxide and formalin fixation. Strongly diffractive crystalline material present in an OSO4 fixed specimen was interpreted as precipitated dye as the observed spotted pattern was much weaker in formalin treated tissue samples. OSO4 easily reacts with ethylenic double bonds. An alternative and more likely explanation therefore is that regular, electron dense crystalline structures are formed due to reaction of OSO4 with a highly organized lattice of unsaturated membrane lipids. 

Fig. 79. Bronchiolar kinocilium showing membrane bound vesicles in the outer zone alongside the axonema. Bronchiolar epithelium of a female rat (breeder Winkelmann, Borchen-Kirchborchen) treated for 5 days per week with 150 mg DL-a-tocopherol acetate per kg body weight x day. The colloidal aqueous solution was injected intramuscularly from April 12 to June 20, 1967 for a total of 45 days. Fixed under methitural anaesthesia by intratracheal instillation of 2.5 % glutaraldehyde in phosphate buffer (pH 7.4) before opening the thorax. After washing in phosphate buffer the tissue was postfixed with 1 % osmium tetroxide in phosphate buffer for 2 h. Contrasted en bloc for 12 h with 0.5 % uranyl acetate in 70 % ethanol. Embedded in a 2 8 mixture of methyl and butyl methacrylate. Sectioned at 50 nm. Lead citrate after Reynolds (1963). Plate 31/12...

ESCA measurements ESCA(Electron Spectroscopy for Chemical Analysis) spectra were obtained on a Shimazu ESCA Model 650-B electron spectrometer using MgKa radiation. Samples of membranes cast from chloroform (Cf) - trifluoro-ethanol (TFE) 10 1 mixture, followed by fixation and staining of membranes by osmium tetroxide (OsO ) were coated onto plane glass plate, and fixed to the probe by doublesided tape. 

Phospholipids occur in cells mostly as components of cellular membranes in which they are in close structural relation to proteins. This interaction is an important factor in relation to the problem of phospholipid preservation during dehydration and embedding of tissues. Data on total phospholipid retention, following labeling with fatty acids are given by Kom and Weisman (1966) in the amoeba and by Dermer (1968) in rat intestine. The former have noted better retention of phospholipids than of neutral lipids, while the latter reported that both were retained to the same extent Very little loss of phospholipids, determined chemically, from erythrocyte stroma fixed with glutaraldehyde and osmium tetroxide in the presence of Ca ions was reported by Mitchell (1969). The loss increased from 2 to 7% if the stroma was stored for 7-10 days prior to fixation. 

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