Detecting Cell Death and Proliferation in Xenopus Embryos

To perform TUNEL and pH3 staining, Xenopus embryos were fixed in MEMFA (4% paraformaldehyde, 0.1 M MOPS pH 7.4, 1 mM MgSO₄, and 2 mM EGTA), washed with PBS, and then bleached in a bleach solution (3% H₂O₂, 5% formamide, and 5X SSC). For TUNEL assays, bleached embryos were end-labeled using digoxigenin-11-dUTP (Sigma-Aldrich) and TdT (Invitrogen, Carlsbad, CA). Labeled ends were detected with alkaline phosphatase-labeled anti-digoxigenin antibodies (1:1000; Roche) and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. For pH3 staining, bleached embryos were blocked in a blocking solution (1% bovine serum albumin and 5% goat serum in PBS) and probed with anti-histone H3 (1:1000; Abcam, Cambridge, UK) and anti-rabbit IgG AP-linked antibody (1:2000; Santa Cruz Biotechnology). pH3-positive cells were detected by nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate [39 (link), 60 (link)].