Effects of different fixatives on the nuclear-cytoplasmic localization of YAP in human corneal epithelial cells under hyperosmotic stress

MethodsImmortalized human corneal epithelial cells were divided into control group and hypertonic group.After 1 day of normal culture, cells of the hypertonic group were exposed to hyperosmotic medium at 450 mOsM by adding sodium chloride for 1 hour.No special treatment was given to the control group.Both groups of cells were fixed with four different fixatives, including 4% paraformaldehyde (PFA), -20 ℃ precooled absolute ethanol, -20 ℃ precooled methanol-acetone 1∶1 mixture, and Zamboni fixative solution for 20 minutes.Subsequent to fixation, immunofluorescent staining procedures were performed to identify the intracellular localization of YAP in the two groups.

Results  After fixation with 4% PFA, human corneal epithelial cells showed normal morphology with YAP mainly in the nucleus in both groups, and there was no significant difference in the mean nuclear YAP fluorescence intensity between the two groups ( t=1.803, P=0.121).After fixation with absolute ethanol, cells showed some degree of shrinkage and deformation, diffuse YAP fluorescence staining with YAP-positive signals mainly localized in the cytoplasm in both groups, and the mean nuclear YAP fluorescence intensity was slightly decreased in the hypertonic group compared with the control group, but the difference was not statistically significant ( t=0.803, P=0.453).After fixation with methanol-acetone 1∶1 mixture, cells were crenulated with YAP mainly in the cytoplasm, and the mean nuclear YAP fluorescence intensity in the hypertonic group was slightly decreased compared with the control group, but the difference was not statistically significant ( t=1.067, P=0.327).After fixation with Zamboni solution, the cell structure was complete and clearly outlined, and the YAP nucleoplasmic translocation phenomenon could be clearly observed in cells in different states.The mean nuclear YAP fluorescence intensity in the hypertonic group was 197.5±34.5, which was significantly higher than 62.2±10.0 in the control group ( t=7.530, P<0.001).

Conclusions  In the immunofluorescence staining experiment, the nucleoplasmic localization of YAP in corneal epithelial cells is affected by different fixative treatments.Zamboni fixative is better than 4% PFA, absolute ethanol, and methanol-acetone 1∶1 mixture in observing nuclear translocation of YAP after hypertonic stimulation.

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