Whole mount in situ hybridization and immunohistochemistry for zebrafish larvae

Abstract

In situ hybridization enables visualization of mRNA localization, and immunohistochemistry enables visualization of protein localization within a tissue or organism. Both techniques have been extensively utilized in zebrafish (Thisse et al., Development 119:1203–1215, 1993; Dutton et al., Development 128:4113–4125, 2001; Gilmour et al., Neuron 34:577–588, 2002; Lyons et al., Curr Biol 15:513–524, 2005) including for visualization of mRNA localization in Schwann cells (Lyons et al., Curr Biol 15:513–524, 2005; Monk et al., Science 325:1402–1405, 2009). For in situ hybridization, here, we outline how to generate RNA probes, conduct whole mount in situ hybridization for larvae, and list RNA probes that label different stages of Schwann cell development in zebrafish. For immunohistochemistry, the protocol we outline can be used to mark Schwann cells of sensory and motor nerves to examine properties such as developmental stage, morphology, proliferation, and apoptosis.