Analysis of Gene Expression by In Situ Hybridization on Adult Zebrafish Brain Sections

INTRODUCTION Whole-mount in situ hybridization is a powerful method for the analysis of gene expression in zebrafish. However, the thickness of adult brain tissue often hampers penetration of reagents, making the direct application of protocols normally used for embryonic stages on adult tissues difficult. This protocol presents a detailed procedure for the dissection, preparation, and processing of the adult zebrafish brain for detection of gene expression in situ using digoxigenin (DIG)-labeled RNA probes on vibratome sections: The entire procedure is presented, including synthesis of labeled antisense RNA, sectioning of the adult zebrafish brain, and in situ hybridization. Using this method, all parts of the tissue are easily labeled and expression patterns are reproduced faithfully between trials. Unlike whole-mount preparations, gene expression examined on vibratome sections can easily be compared to a detailed zebrafish brain atlas and assigned to specific tissues.