In Situ Hybridization of Whole-Mount Mouse Embryos with RNA Probes: Preparation of Embryos and Probes
This protocol was adapted from “Techniques for Visualizing Gene Products, Cells, Tissues, and Organ Systems,” Chapter 16, in Manipulating the Mouse Embryo, 3rd edition, by Andras Nagy, Marina Gertsenstein, Kristina Vintersten, and Richard Behringer. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.INTRODUCTION
This 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 H2O 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.
