Whole-Mount In Situ Hybridization of Short-Tailed Fruit Bat (Carollia perspicillata) Embryos with RNA Probes

Abstract

INTRODUCTIONThis protocol describes whole-mount in situ hybridization of short-tailed fruit bat (Carollia perspicillata) embryos with RNA probes. This technique allows direct visualization of the mRNA expression pattern of a gene of interest within embryos fixed at a wide range of developmental stages. Due to limitations in probe penetration, the size of the specimen must be considered when using whole-mount procedures. In addition, the preparation of probe is an important step in whole-mount in situ hybridization. Two primary considerations for making a good probe are (1) a good template sequence and (2) an efficient labeling reaction. In our experience, the ideal template sequence for a whole-mount in situ probe should be 300-1000 bp in length. We have had little success using shorter probes, although they can be made to work in a case-by-case (and presumably sequence-dependent) manner. Typically, one begins with a cDNA or coding genomic fragment cloned into an in vitro transcription vector. The linearized vector is purified and used as the template for in vitro transcription. In this protocol, the in situ hybridization procedure is presented as a 3-d schedule: Day 1, Pre-treatment and hybridization; Day 2, Post-hybridization washes and antibody incubation; and Day 3, Post-antibody washes and color reaction.