Freeze-Crack Technique to Study Epidermal Development in Zebrafish Using Differential Interference Contrast Microscopy and Fluorescent Markers

The zebrafish is a model organism used to study organogenesis during vertebrate development; however epidermis development has been the focus of only a few studies. Thus, new methodologies to highlight and study epidermal cells could be valuable to deepen our understanding of skin development. Large-scale mutagenic screenings have already identified many zebrafish mutants, which are models for human developmental diseases, however only four epidermis mutants have been isolated. Novel screening techniques are needed to improve this collection. We designed and tested a novel freeze-crack technique to obtain, fix, and stain epidermal cells from 5 days postfertilization zebrafish larvae. Using commercially available fluorescent markers and differential interference contrast (DIC) microscopy, we were able to label and highlight subcellular structures such as microridges, cell boundaries, nuclei, and the Golgi complex from epidermis cells. Acquiring and processing epidermis samples from 15 to 75 larvae takes about 2-4 h, respectively. Therefore this method could be used as part of large-scale screenings. In addition, we present a more extensive protocol for antibody staining, which could be employed for more specific studies.