Whole Mount RNA In Situ Hybridization on Zebrafish Embryos: Hybridization

INTRODUCTION

This protocol describes procedures for detecting gene expression in zebrafish embryos in situ using labeled RNA probes. The labels (digoxigenin or fluorescein) are detected using alkaline phosphatase-conjugated antibodies that catalyze a chromogenic reaction, producing visible blue or red products. Two-color in situ hybridization can be performed by labeling with a combination of digoxigenin- and fluorescein-labeled probes and detecting the digoxigenin and fluorescein with sequential alkaline phosphatase reactions using different chromogenic substrates. In the method described here, the fluorescein-labeled probe is detected with the red substrate, but either probe can be detected with either substrate.