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

Yuan Qin,Wenting Jiang,Meng Gao,Hanyu Liu,Guohua Gong,Anqi Li,Yufei Gao,Xiangang Tian

doi:10.3390/biom11050711

Abstract

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.

Highlights

Mitochondria are major highly dynamic organelles regulated by fission and fusion.Mitochondrial size, shape, and location are variable in different cell types [1,2,3,4]
We first detected the effect of fixative on cell morphology before and after fixation. 95% ethanol (ETHO) caused cells shrinkage after 10 min fixation
The mitochondria within live cells were generally visualized through the mitochondrial dyes that can accumulate into the mitochondrial matrix

Summary

Introduction

Mitochondria are major highly dynamic organelles regulated by fission and fusion.Mitochondrial size, shape, and location are variable in different cell types [1,2,3,4]. Mitochondria are major highly dynamic organelles regulated by fission and fusion. Mitochondria autonomously respond to energy demands and environmental changes in cells by reshaping morphology [4,5,6]. The morphology of mitochondria is directly related to the functions of cells and tissues [7,8,9,10]. Mitochondria in live cells can be directly visualized by microscope technology [11,12]. In some cases, cell samples cannot be imaged immediately after collection, they have to be frozen in a specific state using chemical fixation [13,14]. The preserved mitochondria can be visualized by immunocytochemistry or confocal microscopy

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