Furukawa T, Ohno S, Oguchi H, Hora K, Tokunaga S, Furuta S
Department of Internal Medicine, Shinshu University School of Medicine, Nagano, Japan.
Kidney Int. 1991 Oct;40(4):621-4. doi: 10.1038/ki.1991.253.
The widths of slit diaphragms in rat glomeruli were quantitatively measured on electron micrographs obtained by the rapid-freezing and freeze-substitution (FS) fixation method and compared to those prepared by conventional chemical fixation procedures, such as tannic acid, glutaraldehyde and osmium tetroxide (TGO) or glutaraldehyde and osmium tetroxide (GO) fixation. When fixed by FS using 2% osmium tetroxide, glomerular slit diaphragms had widths of 33.8 +/- 3.6 nm, but when fixed by TGO or GO they had widths of 47.0 +/- 4.3 and 48.7 +/- 5.5 nm, respectively. The width of slit diaphragms was significantly narrower after cryofixation than after conventional TGO or GO fixation (P less than 0.001). Similarly, the distance between neighboring foot processes at 50 nm from slit diaphragms was significantly narrower after cryofixation (P less than 0.001). The widths were 98.6 +/- 17.1 nm after TGO fixation, 122.0 +/- 29.3 nm after GO fixation and only 54.8 +/- 18.7 nm after FS fixation. These foot processes were significantly broader after FS fixation. The foot processes were 179.2 +/- 59.4 nm in breadth after TGO fixation (P less than 0.001), 188.8 +/- 78.2 nm after GO fixation (P less than 0.01) and 235.1 +/- 87.2 nm after FS fixation. These results suggest that the cytoplasm of foot processes shrinks when the tissue is prepared by conventional chemical fixation methods, and that the broadening of slit diaphragms is an artifact separation of the margins of adjacent foot processes due to shrinkage. We propose that TGO fixation alters the structure of slit diaphragms by causing them to shrink.
采用快速冷冻和冷冻置换（FS）固定方法获取电子显微镜图像，对大鼠肾小球裂孔隔膜的宽度进行定量测量，并与采用常规化学固定程序（如单宁酸、戊二醛和四氧化锇（TGO）或戊二醛和四氧化锇（GO）固定）制备的样本进行比较。使用2%四氧化锇通过FS固定时，肾小球裂孔隔膜的宽度为33.8±3.6nm，但采用TGO或GO固定时，其宽度分别为47.0±4.3nm和48.7±5.5nm。冷冻固定后裂孔隔膜的宽度明显窄于传统TGO或GO固定后（P<0.001）。同样，冷冻固定后，距离裂孔隔膜50nm处相邻足突之间的距离也明显变窄（P<0.001）。TGO固定后宽度为98.6±17.1nm，GO固定后为122.0±29.3nm，而FS固定后仅为54.8±18.7nm。FS固定后这些足突明显更宽。TGO固定后足突宽度为179.2±59.4nm（P<0.001），GO固定后为188.8±78.2nm（P<0.01），FS固定后为235.1±87.2nm。这些结果表明，采用传统化学固定方法制备组织时，足突的细胞质会收缩，裂孔隔膜变宽是相邻足突边缘因收缩而人为分离的结果。我们认为TGO固定通过使裂孔隔膜收缩而改变其结构。
