IMPROVED PRESERVATION OF THE SUBEPIDERMAL EXTRACELLULAR-MATRIX IN AXOLOTL EMBRYOS USING ELECTRON-MICROSCOPIC TECHNIQUES BASED ON CRYOIMMOBILIZATION

The purpose of this methodological survey was to find optimal methods for the fixation and demonstration of glycosaminoglycans, mainly hyalu ronan, and proteoglycans, in subepidermal extracellular matrix (ECM) r egions of axolotl embryos, We compared Living ECM in the laser scannin g microscope (LSM) with chemically fixed or cryoimmobilized extracellu lar matrix in the transmission (TEM) and scanning electron microscope (SEM), The gel-like structure of living extracellular matrix in the LS M undoubtedly provides the most natural state, whereas shrinkage of th e extracellular matrix occurs during conventional fixation and dehydra tion for TEM or SEM, Among the methods used for fixation and processin g of subepidermal extracellular matrices for SEM, plunge-freezing/free ze-drying is to be preferred, Still more satisfying, however, are resu lts 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 adeq uately frozen. Conversely, the yield in adequately frozen specimens wi thout cryoprotection was insufficient, In these specimens, the ECM con tained honeycomb-like structures which, in the current literature, are regarded as hyaluronan. (C) 1997 Academic Press.