Ii 116b3

Whole-mount immunofluorescent staining was performed as previously reported (Ning et al., 2013 (link)). Briefly, embryos were fixed with 4% phosphate-buffered paraformaldehyde and washed with 0.3% Triton X-100 and 0.1% Tween-20 in PBS for 20 min before immunostaining. The embryos were stained with the indicated antibodies, including anti-GFP (1/1000; A-11122, Invitrogen), anti-GFP (1/1000; A-11120, Invitrogen), anti-Collagen type II (Col2) (1/100; II-116B3, Developmental Studies Hybridoma Bank), anti-pH3 (1/1000; 3377, Cell Signaling Technology) and anti-p-Smad1/5/8 (1/200; 9511, Cell Signaling Technology). All immunofluorescent images were captured using a Nikon A1R+ confocal microscope with the same settings for all experiments.
TUNEL assays were performed using the In Situ Cell Death Detection Kit, TMR red (12156792910, Roche) according to the manufacturer's instructions. DAPI was used to visualize nuclei.

Embryos fixed in 4% PFA were permeabilized with 0.1% Triton X‐100, rinsed several times in PBST buffer, and then blocked in 5% goat serum. For Col2 immunostaining, anti‐Collagen type II (1:100; II‐116B3, Developmental Studies Hybridoma Bank) and Cy3‐goat anti‐mouse (A0521, Beyotime) were used as the primary and secondary antibodies, respectively.⁵ For phosphohistone‐H3 (PH3) immunostaining, pH3‐Ser‐10 (sc‐8656‐R, Santa Cruz) and Cy3‐goat anti‐rabbit (A0516, Beyotime) were used as the primary and secondary antibodies, respectively. For TUNEL staining, the in situ Cell Death Detection Kit (12156792910, Roche) was used in accordance with the manufacturer's instruction. Images were captured using an FV1000 Bx61 or SpinSR IX83 confocal microscope (Olympus). At least five embryos from different treatments were used for quantification and statistical analysis.