engleski

Optimized protocol for whole-mount immunohistochemistry in zebrafish embryos (Danio rerio)

In the last couple of decades, the use of zebrafish model in biomedical and toxicological research is exponentially increasing. Many studies investigate the physiological/ecotoxicological function of different proteins localized across different zebrafish organs/tissues. Whole-mount preparations coupled with fluorescence microscopy are widely used for this task. To date there is no single approach for conducting whole-mount immunohistochemistry in zebrafish embryos. In this study we used zebrafish embryos (96 hpf) to determine key parameters in preparation of zebrafish embryos for the use in whole-mount immunohistochemistry using two antibodies detecting Na+/K+ATPase and ZN-8 membrane proteins. Two fixatives (4% paraformaldehyde/4% PFA and Dents fixative) and three different incubation temperatures (37 °C/30 min, 70 °C/15 min, 95 °C/5 min) were tested to optimize the whole-mount immunohistochemistry approach. Results showed that the use of Dents fixative compared to 4% PFA fixative enables better binding of the tested primary antibodies and reveals the known localization of Na+/K+ATPase and ZN-8 proteins in zebrafish embryos. However, the use of different incubation temperatures was not crucial for the binding of the tested antibodies. Overall, this work presents a unified approach for whole-mount immunohistochemistry that can be used to test optimal conditions for localization of proteins in zebrafish embryos.

whole-mount ; immunohistochemistry ; zebrafish ; optimization

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