Yoshimura A, Ohno S, Nakano K, Oniki H, Inui K, Ideura T, Koshikawa S
Department of Internal Medicine, Showa University, Fujigaoka Hospital, Yokohama, Japan.
Histochemistry. 1991;96(2):107-13. doi: 10.1007/BF00315980.
The ultrastructure of anionic sites in the lamina rara externa (LRE) of rat glomerular basement membrane (GBM) was studied in three dimensions by a quick-freezing and deep-etching method using polyethyleneimine (PEI) as a cationic tracer. Results were compared with those obtained with conventional ultrathin sections examined by transmission electron microscopy. Examination with the quick-freezing and deep-etching method was done without (group 1) or with (group 2) contrasting/fixation with a phosphotungstic acid and glutaraldehyde mixture and post-fixation with osmium tetroxide, which were necessary for visualization of PEI particles by conventional ultrathin sections. Using the quick-freezing and deep-etching method without following contrasting/fixation and post-fixation (group 1), many PEI particles were observed to decorate around fibrils, which radiated perpendicularly from the lamina densa to connect with the podocyte cell membrane. The arrangement of PEI particles was not as regular as that previously reported using conventional ultrathin sections. In contrast, the tissue that was studied with quick-freezing and deep-etching followed by contrasting/fixation and post-fixation (group 2) showed a shrunken appearance. The arrangement of PEI particles was regular (about 20 particles/1000 nm of LRE) as that previously observed using conventional ultrathin sections. However, the number of PEI particles on the LRE was markedly decreased and interruption of decorated fibrils was prominent, as compared with group 1. Ultrastructural examination using conventional ultrathin sections with contrasting/fixation and post-fixation (group 3) demonstrated PEI particles on the LRE in reasonable amounts (18-21 particles/1000 nm of LRE) with fairly regular interspacing (45-65 nm) as reported previously.(ABSTRACT TRUNCATED AT 250 WORDS)
采用聚乙烯亚胺（PEI）作为阳离子示踪剂，通过快速冷冻和深度蚀刻法对大鼠肾小球基底膜（GBM）外疏松层（LRE）中阴离子位点的超微结构进行了三维研究。将结果与通过透射电子显微镜检查的传统超薄切片所获结果进行比较。采用快速冷冻和深度蚀刻法进行检查时，一组（第1组）未使用磷钨酸和戊二醛混合物进行反差染色/固定以及四氧化锇后固定，而另一组（第2组）则进行了上述处理，这些处理对于通过传统超薄切片观察PEI颗粒是必要的。采用快速冷冻和深度蚀刻法且未进行后续反差染色/固定及后固定（第1组）时，观察到许多PEI颗粒附着在从致密层垂直放射状延伸并与足细胞细胞膜相连的纤维周围。PEI颗粒的排列不像先前使用传统超薄切片报道的那样规则。相比之下，经过快速冷冻和深度蚀刻后再进行反差染色/固定及后固定的组织（第2组）呈现出收缩的外观。PEI颗粒的排列与先前使用传统超薄切片观察到的一样规则（约20个颗粒/1000 nm的LRE）。然而，与第1组相比，LRE上的PEI颗粒数量明显减少，且被标记纤维的中断现象突出。使用经过反差染色/固定及后固定的传统超薄切片进行超微结构检查（第3组）显示，LRE上的PEI颗粒数量适中（18 - 21个颗粒/1000 nm的LRE），间距相当规则（45 - 65 nm），与先前报道一致。（摘要截短于250字）
