Improved preservation of the subepidermal extracellular matrix in axolotl embryos using electron microscopical techniques based on cryoimmobilization.

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.