Preservation of Erythrocyte Ghost Ultrastructure Achieved by Various Fixatives

We have evaluated the quality of ultrastructural preservation of erythrocyte ghosts achieved under various electron microscopic preparative conditions. Initially, negative staining was used to monitor gross morphology of the ghosts. Of several negative stains used (phosphotungstate, silicotungstate, ammonium molybdate, and uranyl acetate), all but uranyl acetate resulted in fragmentation of membranes and the appearance of small vesicular structures. Further, preservation of gross membrane ultrastructure was greatly enhanced when samples were fixed with either 4.0-5.0% glutaraldehyde or 1% osmium tetroxide (OsO(4)) before they were stained with uranyl acetate. We then examined the ability of these fixatives to preserve membrane fine structure, as monitored by thin-sectioning procedures. In these studies, fixation with 1% osmium tetroxide (alone or in conjunction with 5% glutaraldehyde) resulted in a trilamellar image about 95 A in width. Fixation with 5% glutaraldehyde alone provided a markedly different result. The membrane now appeared as a single line about 160 A wide with regions of varying electron density throughout. This result suggests that glutaraldehyde used alone may reveal the location of membrane proteins that are obscured or removed by OsO(4) fixation. This point would seem to be supported by the results obtained when erythrocyte membranes were extracted with 5 mM EDTA after fixation in either 5% glutaraldehyde or 1% OsO(4). While only 10% of the detectable protein was solubilized from glutaraldehyde-treated erythrocyte membranes, 85% was solubilized from OsO(4)-treated ghosts. Among these latter proteins are three that migrated on Ouchterlony double-diffusion agar plates at the same position as three known proteins with molecular weights of about 200,000. Additional studies indicated that, even during a routine pre-embedding procedure, OsO(4) led to solubilization of as much as 8 times the amount of protein as glutaraldehyde alone. Although the erythrocyte membrane has a notoriously weak association with its proteins, we feel that our studies provide a cautionary note with regard to the use of OsO(4) as a fixative in other membrane systems.