Alkaline phosphatase labeled anti digoxigenin antibody

Two-cell and eight-cell stage embryos were unilaterally injected with kdm5c MO and fixed at appropriate stages in MEMFA (4% paraformaldehyde, 0.1 M MOPS pH 7.4, 1 mM MgSO₄, and 2 mM EGTA) overnight at 4 °C and then dehydrated in 100% methanol prior to storage at − 20 °C. To prepare the antisense digoxigenin-labeled probes, DNA templates were linearized using restriction enzymes. Probes were generated using SP6 or T7 RNA polymerase (Ambion). Probes were detected using alkaline phosphatase-labeled anti-digoxigenin antibodies (1:1000; Roche, Basel, Switzerland) and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate [59 (link)].

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)].

The LacZ and white probes were generated by transcription from linearized pBluescript template plasmids (kindly provided by Dr. Mattias Mannervik) with T3 or T7 RNA polymerase (Thermo Fisher) and Dig RNA labelling mix (Roche) according to the manufacturer. Embryos were aged and fixed with 9% formaldehyde in fixation buffer (1.3x PBS, 67 mM EGTA, pH 8) for 25 min. For in situ hybridization, fixated embryos were permeabilized with xylene and rehydrated as well as postfixated with 5% formaldehyde in PBT (1x PBS, 0.1% Tween-20) for 25 min. Embryos were treated with proteinase K (4 µg/ml) for 8 min, followed by another round of postfixation for 25 min, before hybridization with the probes at 55 °C for overnight in hybridization buffer (50% formamide, 5x SSC, 100 µg/ml sonicated boiled ssDNA, 0.1% Tween-20). Samples were incubated with alkaline-phosphatase-labeled anti-digoxigenin antibody (1:2000, Roche) overnight at 4 °C, and developed with 0.6 mg/ml nitrotetrazolium blue chloride (NBC) and 0.3 mg/ml 5-bromo-4-chloro-3-indolyl phosphate disodium salt (BCIP). Samples were dehydrated by repeated washes in ethanol, rinsed in xylene, and mounted in Permount (Fisher).