Whole-Mount in situ Hybridization for Xenopus Nrf2

Nrf2 sequences were retrieved from the NCBI and Xenbase (http://www.xenbase.org, accessed on 9 February 2022) databases. Following PCR amplification, fragments of Nrf2 were cloned into the PCS107 plasmid. The plasmids were then linearized using BamHI and the mMACHINE SP6 Transcription Kit (Thermo Fisher, Waltham, MA, USA) was used to synthesize the DIG-labeled antisense fragment. Three normal live embryos in the control and 25 μmol/L groups were randomly selected from each dish for WISH at stages 19, 32, and 38 (nine samples per treatment group per stage). Then, the embryos were fixed in MEMFA (0.1 mol/L MOPS, 2 mmol/L EGTA, 1 mmol/L MgSO₄, 3.7% formaldehyde, pH 7.4) for 1–2 h at room temperature and completely dehydrated using different grades of absolute ethanol. WISH was performed according to a previously described procedure [27 (link)]. Alkaline phosphatase (AP)-coupled anti-DIG antibody was used to recognize the DIG-labeled probe. Staining was conducted in BM Purple (Roche) at 37 °C. The embryos were observed under a stereomicroscope (SteREO Discovery. V8, Carl Zeiss, Jena, Germany), and the images were taken using a digital camera (AxioCam MRc, Carl Zeiss, Jena, Germany).