A microscopic survey on the efficiency of well-known routine chemical fixatives on cryosections

This study was designed to analyze and compare tissue preservation efficiency of acetone (AC), formaldehyde (FA) and paraformaldehyde (PFA) on cryosections. Brain, kidney, heart and liver tissue of adult Balb/c mice were fixed with either FA or PFA prior to cryosectioning, or fixed with AC alone immediately after cryosectioning. Hematoxylin and eosin staining showed that AC is a poor fixative in preserving the general tissue and cellular organization. PFA, and to a lesser extent FA, produced significantly better results. Another set of cryosections were further analyzed to test the properties of those fixatives to preserve proteins from specific cell structures. Cytokeratin filaments, F-actin filaments and nuclei were immunolabeled and examined using confocal microscopy. Results demonstrated that, overall, PFA is the best fixative tested. However, FA fixation gave poor results in preserving neuronal tissues. Immunofluorescence confirmed the inefficiency of AC fixation, after which no specific labelling of cytokeratin filaments was detectable. Nevertheless, actin filaments were detectable on AC-fixed samples, a finding that was supported by the quantification of fluorescein-phalloidin binding to F-actin. Overall, the data suggest that AC fixation is unacceptable for preservation of most samples, whereas FA and PFA fixation should be chosen according to the tissues and proteins to be studied.