Anti ph3

To visualize the apical junctional meshwork (Figure 1C), cerebral walls were fixed in 4% paraformaldehyde prepared in phosphate buffer (pH 7.4) and subject to whole-mount staining with anti–ZO-1 mouse monoclonal antibody (33-9100, Thermo Fisher Scientific), followed by Alexa Fluor 488–labeled anti–mouse IgG antibody (A-11029, Thermo Fisher Scientific). To determine the VZ identity of dissociated cells subjected to AFM measurement or live-cell imaging for volume measurement (Figure 7), they were fixed with 4% paraformaldehyde in phosphate buffer (pH 7.4) and immunostained with anti-Sox2 rabbit polyclonal antibody (ab97959, Abcam), followed by Alexa Fluor 488–labeled anti–rabbit IgG antibody (A-11008, Thermo Fisher Scientific). To determine the density of M-phase cells at the apical surface, cerebral walls treated with DMSO, blebbistatin, Y-27632, or nocodazole were coronally frozen-sectioned (16 μm) and immunostained with anti-pH3 (rabbit, 06-570, MILLIPORE, 1:300).

Zebrafish embryos were fixed overnight in 4% paraformaldehyde in PBS at 4 °C. For histology, 5 µm thin paraffin sections were cut and stained with haematoxylin and eosin. Embryos were fixed for antibody staining with 4% PFA and whole-mount immunohistochemistry was performed according to Dolez et al. [20] (link), using the following primary antibodies: rabbit polyclonal anti-PH3 (1∶1000; Millipore); mouse monoclonal anti-Pax7 (1∶20; Hybridoma Bank), mouse monoclonal anti-F59 (1∶100; Hybridoma Bank); mouse monoclonal anti-F310 (1∶100, Hybridoma Bank); rabbit polyclonal anti-Laminin (1∶400; Sigma). The following secondary antibodies were used: Alexa Fluor 488 goat anti-mouse IgG1(y1) (A-21121, Invitrogen); Alexa Fluor 594 goat anti-rabbit IgG (H+L) (A-11012, Invitrogen). E13.5 mouse embryos were fixed in 4% paraformaldehyde, de-hydrated and included in paraffin. Haematoxylin-eosin staining was performed following standard protocols. Images were detected using a Zeiss Axioplan microscope equipped with a Leica DC500 digital camera.
Phospho-H3 immunostaining was quantified by counting positive cells present in the same six somites of the trunk region in ten embryos of each category.

Cells were fixed with 70% ethanol while gently vortexing. The fixed cells were permeabilized with 0.25% Triton X-100 in PBS on ice for 15 min. The cells were incubated with anti-pH3 (Millipore, Burlington, MA, USA) antibody for 2 h, and then incubated with the corresponding secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) at room temperature in the dark for 1 h. Cells were incubated with DNase-free RNase A at 37 °C for 30 min and then with propidium iodide (PI) at 37 °C in the dark for another 30 min. The percentage of pH3-positive cells was determined by flow cytometry [43 (link)].

Fly tissues were dissected in PBS and fixed for 30 min in 4% formaldehyde. Blocking and antibody incubations were performed in PBT (0.3% BSA, 0.3% Triton X-100 in PBS). Primary and secondary antibodies were incubated overnight at 4 °C in PBT. The following primary antibodies were used: anti-Cathepsin-L (Abcam ab58991, 1:400), anti-GABARAP (Cell Signaling Technology #13733, 1:400), anti-pH3 (Millipore #06-570, 1:1000), anti-Kenny^{23 (link)} (gift from Dr N. Silverman, 1:600), anti-Relish (#Abin1111036, RayBiotech 130-10080, 1:300). Specificity of the anti-Relish antibody was tested using Relish E20 mutants^{63 (link)} (Supplementary Fig. 18). Washes were performed in PBW (0.1% Tween-20 in PBS). All images were acquired using Carl Zeiss LSM710 or LSM880 confocal microscopes, using a ×63 Apochromat objective. Images were post-processed in Fiji for co-localization studies.