In situ hybridization of whole-mount mouse embryos with RNA probes: preparation of embryos and probes.

INTRODUCTIONThis is the first of two protocols describing how to perform in situ hybridization on whole mouse embryos. Here we describe how to fix and permeabilize embryos with detergent and how to synthesize single-stranded RNA probes (riboprobes) in vitro using UTP labeled with the plant steroid digoxigenin (DIG-11-UTP). Antisense digoxigenin-labeled riboprobes are synthesized as run-off transcripts from linearized plasmid templates using bacteriophage RNA polymerases (T3, T7, SP6) under standard conditions. The probe is precipitated with ethanol in the presence of NaCl, and after resuspension in DEPC-treated H(2)O is stable at -20°C for many months. Probes ranging from 250 to 1500 bp have been used successfully. The optimal probe concentration is in the 100 ng to 1 μg/mL range and should be determined empirically.