engleski

Improved histological approach for localization and expression analysis of (non)membrane proteins in zebrafish cryosections

Ever increasing use of zebrafish model for studying different biomedical and toxicological issues linked to the physiological/ecotoxicological function of various (non)membrane proteins, makes development and/or optimizing existing methods for studying protein localization/expression in this model extremely important. Compared to whole-mount preparations and/or parafin-embedded tissue sections, zebrafish cryosections are still poorly used in this context. In this study, we used zebrafish embryo/larvae (24-120 hpf; N=20 individuals/group) to: 1) determine key factors for preparation of high-quality zebrafish cryosections, 2) optimize the protocol for localization/expression of a nonmembrane F-actin protein using fluorescently-labeled phalloidin (FITC-Phalloidin), 3) determine the use of optimized protocol for immunofluorescence analyses of Na+/K+ATPase membrane protein, and 4) investigate stage development-related changes in F-actin and Na+/K+ATPase protein expression. The results revealed the highest quality of zebrafish cryosections after samples underwent 1 h-fixation in 4 % paraformaldehyde (PFA), incubation in 2.5 % bovine gelatine/25 % succrose mixture, followed by cutting to 8 μm thickness at 20 °C. Fluorescence microscopy analysis of phalloidin-labeled zebrafish skeletal muscles revealed that 1 h-4% PFA fixed samples enabled optimal phalloidin-to-F-actin binding. Further (immuno)fluorescence analyses revealed detailed localization of F-actin and Na+/K+ATPase in different zebrafish organs/tissues and their respective stage-dependent expression increase in somitic muscles and pronephros. Finally, staining in zebrafish cryosections and whole-mount samples revealed organ-specific and zone-dependent localizations of Na+/K+ATPase α1-subunit. Project financed by Croatian Science Foundation, DANIOTRANS (HRZZ-IP-2019-04-1147).

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