The Combination of Paraformaldehyde and Glutaraldehyde Is a Potential Fixative for Mitochondria.

Mitochondria are highly dynamic organelles, constantly undergoing shape changes, which are controlled by mitochondrial movement, fusion, and fission. Mitochondria play a pivotal role in various cellular processes under physiological and pathological conditions, including metabolism, superoxide generation, calcium homeostasis, and apoptosis. Abnormal mitochondrial morphology and mitochondrial protein expression are always closely related to the health status of cells. Analysis of mitochondrial morphology and mitochondrial protein expression in situ is widely used to reflect the abnormality of cell function in the chemical fixed sample. Paraformaldehyde (PFA), the most commonly used fixative in cellular immunostaining, still has disadvantages, including loss of antigenicity and disruption of morphology during fixation. We tested the effect of ethanol (ETHO), PFA, and glutaraldehyde (GA) fixation on cellular mitochondria. The results showed that 3% PFA and 1.5% GA (PFA-GA) combination reserved mitochondrial morphology better than them alone in situ in cells. Mitochondrial network and protein antigenicity were well maintained, indicated by preserved MitoTracker and mitochondrial immunostaining after PFA-GA fixation. Our results suggest that the PFA-GA combination is a valuable fixative for the study of mitochondria in situ.