Cell membrane osmium tetroxide fixative

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. 

Fig. 85. Bronchiolar ciliated epithelial cell showing membrane bound vesicles between the axonema and the ciliary membrane. Lung of a female rat (No. 4 breeder Winkebnann, Borchen-Kirchborchen) which had inhaled 10 mg powdered Grangesberg magnetite/m3 4 h per day, 5 days per week from August 24 to October 19, 1967 for a total of 40 days. Fixed on January 15, 1968 under methitural anaesthesia by intratracheal instillation of 2.5 % glutaraldehyde in phosphate buffer (pH 7.4) before opening the thorax. Postfixation with 1 % osmium tetroxide in phosphate buffer (pH 7.4). 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). Film 33131...

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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