Phospholipids osmium tetroxide

See review of Williams (25) for retention of lipids with osmium tetroxide generally high rates of phospholipids are retained. 

Phospholipids of the composition present in cells spontaneously form sheetlike phospholipid hilayers, which are two molecules thick. The hydrocarbon chains of the phospholipids in each layer, or leaflet, form a hydrophobic core that is 3-4 nm thick in most biomembranes. Electron microscopy of thin membrane sections stained with osmium tetroxide, which binds strongly to the polar head groups of phospholipids, reveals the bilayer structure (Figure 5-2). A cross section of all single membranes stained with osmium tetroxide looks like a railroad track two thin dark lines (the stain-head group complexes) with a uniform light space of about 2 nm (the hydrophobic tails) between them. 

Electron Microscope Studies. Electron microscope studies of phospholipids have been made by many investigators, but in general the material used has been natural phospholipid and particularly egg yolk phosphatidylcholine. Recently we have studied some pure phosphatidyletha-nolamines (8). Fully saturated phospholipids are crystalline at room temperature and show no interaction with the material commonly used for staining purposes—i.e., osmium tetroxide. If these phospholipids are... 

The effect of double fixation on the extraction of lipids during dehydration and infiltration was studied by Carlier (1968) in rat intestine. When partial dehydration was used according to Idelman (1964, 1965), double fixation (glutaraldehyde followed by osmium tetroxide) reduced the loss of label of 26.6%, encountered with tissues fixed in osmium tetroxide alone, to 12.2%. This improvement was not noted when the tissues were subjected to complete dehydration, which included 100% ethanol and propylene oxide. Another point evaluated in that study was the effect of temperature on the procedure of preparation. It became apparent that when isolated labeled chylomicrons are subjected to partial dehydration the loss of label into the infiltrating resin is reduced from 20.5% at 25° to 11.5% at 4°. Relatively low extraction of lipids from rat intestine labeled with linoleic acid was reported by Buschmann and Taylor (1968) during dehydration in graded ethanols at 0°, and considerably more label was lost to propylene oxide and propylene oxide Araldite mixture, as the last steps were carried out at room temperature. Another study dealing with retention of lipids by the intestine (Dermer, 1968) showed that neutral lipids and phospholipids were extracted to the same extent, and the total loss amounted to about 16%. 

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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