Quick freezing followed by substitution fixation may stop all molecular movements and thus preserve all molecules in the cell as in the living condition. Therefore, it might be said that among many fixations, freeze substitution is ideal for histochemistry. In my laboratory, freeze substitution was first applied to electron microscopical enzyme histochemistry, and it proved able to preserve enough enzyme activities on an excellent ultrastructure by using glutaraldehyde and acrolein as freeze substitution fixatives.
The combination of glutaraldehyde and acrolein with substitution fixation was histochemically successful. As the pure morphology of freeze substitution fixation gains high contrast on biological membranes with osmium tetroxide, attempts were made to investigate the process and the propriety of glutaraldehyde and acrolein in freeze substitution fixation and compared with immersion fixation of conventional chemical fixation. Explorations were also made to get better contrast for the visualization of enzyme localization by electron microscopy. As the glutaraldehyde and acrolein in longer fixation time at room temperature showed enough enzyme activities, freeze substitution enzyme histochemistry could be a reliable method for enzyme histochemistry, allowing us to see the actual site of enzyme action close to the living state.