Whole-Mount Multicolor Fluorescent Labeling by In Situ Hybridization in Astyanax mexicanus Embryos and Larvae

Gene expression analyses by molecular histology are a crucial step in understanding gene function in any model organism. In the teleost Astyanax mexicanus, here we describe in detail the method we have developed to perform double fluorescent in situ hybridization on whole-mount samples. As an illustration, in the result section, we present an analysis of the expression patterns of four mRNAs expressed in the hypothalamus of the surface and cave morphs of A. mexicanus at 3.5 days postfertilization, three neuropeptides (npy, pomca, agrp) and one transcription factor (isl1). Confocal imaging after fluorescent in situ hybridization allows counting cells in distinct but closely related hypothalamic areas. The step-by-step protocol and the comprehensive table of reagents presented here will allow researchers to analyze gene expression in different structures and at various stages, from embryos to older larvae.