Epperlein H H, Schwarz H, Piendl T, Löfberg J, Studer D, Spring H, Müller M
Institut für Anatomie, Techn. Universität, Dresden, Germany.
J Struct Biol. 1997 Feb;118(1):43-61. doi: 10.1006/jsbi.1996.3838.
The purpose of this metholdological survey was to find optimal methods for the fixation and demonstration of glycosaminoglycans, mainly hyaluronan, and proteoglycans, in subepidermal extracellular matrix (ECM) regions of axolotl embryos. We compared living ECM in the laser-scanning microscope (LSM) with chemically fixed or cryoimmobilized extracellular matrix in the transmission (TEM) and scanning electron microscope (SEM). The gel-like structure of living extracellular matrix in the LSM undoubtedly provides the most natural state, whereas shrinkage of the extracellular matrix occurs during conventional fixation and dehydration for TEM or SEM. Among the methods used for fixation and processing of subepidermal extracellular matrices for SEM, plunge-freezing/freeze-drying is to be preferred. Still more satisfying, however, are results obtained with high-pressure frozen/freeze-substituted ECM material in the TEM, for which 10% polyvinyl pyrrolidon +7% methanol was used as a cryoprotectant before high-pressure freezing. In these specimens, no freeze-damage could be observed and they could be regarded as adequately frozen. Conversely, the yield in adequately frozen specimens without cryoprotection was insufficient. In these specimens, the ECM contained honeycomb-like structures which, in the current literature, are regarded as hyaluronan.
本方法学调查的目的是找到固定和显示蝾螈胚胎表皮下细胞外基质（ECM）区域中糖胺聚糖（主要是透明质酸）和蛋白聚糖的最佳方法。我们将激光扫描显微镜（LSM）下的活细胞外基质与透射电子显微镜（TEM）和扫描电子显微镜（SEM）下化学固定或冷冻固定的细胞外基质进行了比较。LSM下活细胞外基质的凝胶状结构无疑提供了最自然的状态，而在用于TEM或SEM的传统固定和脱水过程中，细胞外基质会发生收缩。在用于固定和处理表皮下细胞外基质以进行SEM的方法中，速冻/冷冻干燥是首选。然而，在TEM中使用高压冷冻/冷冻置换的ECM材料获得的结果更令人满意，在高压冷冻前，10%聚乙烯吡咯烷酮+7%甲醇被用作冷冻保护剂。在这些标本中，未观察到冷冻损伤，可认为它们已充分冷冻。相反，没有冷冻保护剂时充分冷冻标本的产量不足。在这些标本中，ECM含有蜂窝状结构，在当前文献中，这些结构被认为是透明质酸。
