Xenopus Embryo Whole-Mount In Situ Hybridization

gpx1 and prdx2 MOs were injected into one or both blastomeres of two cells staged with Xenopus embryos individually, and the MO-injected embryos at the desired stage were fixed in MEMFA (4% paraformaldehyde, 0.1-M MOPS (pH7.4), 1-mM MgSO4, 2-mM EGTA) overnight at 4 °C and then dehydrated before storage in 100% methanol at −20 ℃. To prepare the antisense DIG-labeling probes of hba3 (Accession no. NM_001086328), mpo (Accession no. NM_001087639), and cryba1 (Accession no. NM_001094493), DNA templates were linearized using appropriate restriction enzymes. The probes were synthesized using SP6 or T7 RNA polymerase (Ambion) and were detected using an alkaline phosphatase-labeled anti-digoxigenin antibody (1:1000, Roche, Basel, Switzerland) and NBT/BCIP (nitro blue tetrazolium/5-bromo-4-chloro-3indolyl phosphate) [27 (link)].