McMillan P N, Luftig R B
Proc Natl Acad Sci U S A. 1973 Nov;70(11):3060-4. doi: 10.1073/pnas.70.11.3060.
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.
我们评估了在各种电子显微镜制备条件下获得的红细胞血影超微结构保存的质量。最初，使用负染色来监测血影的总体形态。在所使用的几种负染色剂（磷钨酸盐、硅钨酸盐、钼酸铵和醋酸铀酰）中，除醋酸铀酰外，其他所有染色剂都会导致膜的碎片化以及小泡状结构的出现。此外，当样品在用醋酸铀酰染色之前先用4.0 - 5.0%的戊二醛或1%的四氧化锇（OsO₄）固定时，总体膜超微结构的保存得到了极大增强。然后，我们通过超薄切片程序监测这些固定剂保存膜精细结构的能力。在这些研究中，用1%的四氧化锇（单独或与5%的戊二醛结合）固定会产生宽度约为95埃的三层图像。单独用5%的戊二醛固定则产生了明显不同的结果。此时膜呈现为一条约160埃宽的单线，各处电子密度不同。这一结果表明，单独使用戊二醛可能会揭示被OsO₄固定掩盖或去除的膜蛋白的位置。当红细胞膜在5%的戊二醛或1%的OsO₄中固定后用5 mM的EDTA提取时所获得的结果似乎支持了这一点。虽然从戊二醛处理的红细胞膜中仅可检测到10%的蛋白质被溶解，但从OsO₄处理的血影中85%的蛋白质被溶解。在这些后者的蛋白质中有三种在欧氏双扩散琼脂平板上的迁移位置与三种已知分子量约为200,000的蛋白质相同。进一步的研究表明，即使在常规的包埋前程序中，OsO₄导致溶解的蛋白质量是单独使用戊二醛时的8倍之多。尽管红细胞膜与其蛋白质的结合 notoriously weak（此处可能有误，推测可能是“众所周知地弱”），但我们认为我们的研究为在其他膜系统中使用OsO₄作为固定剂提供了一个警示。
