In Situ Hybridization with 35S-Labeled Probes for Retinoid Receptors

In situ hybridization (ISH) is a technique that enables one to localize an endogenous mRNA in histologtcal and cytological samples to the resolution of nearly single cells. For this reason, ISH is widely used in the fields of genetics, developmental biology, and neurobiology, as well as for dtagnostic purposes in medicine. Our laboratory has established conditions for ISH using ³⁵ S-labeled antisense-RNA probes (riboprobes), whtch allow the specific detection of each type of murine retinoic acid receptor (RAR), retinoid X receptor (RXR), cellular retinol-binding protein (CRBP), and cellular retinotc acid binding protein (CRABP) gene transcripts. ISH studies have thus revealed that most of the retinoid receptor genes have complex and distinct expression patterns during mouse development (1 –6 ). The protocol described hereafter has been inspired by the pioneermg work of Angerer and colleagues (7 ). It has been designed for histological sections of mouse embryos or adult ttssues, but can be applied to samples from other species, including human. Nevertheless, slightly distinct ISH protocols have been described for the detection of RAR transcripts in chick (8 , 9 ) and Xenopus embryos (10 ).