Manual and automated whole-mount in situ hybridization for systematic gene expression analysis in embryonic zebrafish forebrain

Abstract

Large-scale data collections of gene expression patterns have been assembled to construct molecular atlases of embryonic and adult vertebrate brains. These open data collections are proving useful as profound resources for developmental, physiological, and functional studies and provide a basis for deciphering the molecular anatomies of vertebrate brains. The interrogation of gene expression data from such resources allows identifying genes with restricted regional patterns and neuronal profiles. The corresponding cDNA/ EST clones can be ordered from publicly accessible clone collections strongly facilitating the systematic analysis of gene expression. However, in order to characterize the expression of a large number of genes, it is necessary to develop automated procedures that allow high-throughput analysis of samples. In this chapter, we describe our approach to analyze developmental regulatory genes and neuronal markers in the embryonic zebrafish brain by chromogenic whole-mount in situ hybridization (WISH) using manual and automated procedures. For automation, we make use of the InsituPro robot, which enables automated processing of up to 96 samples in parallel. We provide detailed step-by-step protocols of embryo collection, fixation and permeabilization, cDNA template and RNA probe preparation, manual and automated in situ hybridization, immunohistochemical detection and staining, and mounting and imaging of successfully stained specimen.