Anti p h3s10

Primary antibodies used were: anti-insulin (Catlog#14976982, IF/IHC, 1:200 dilution) and anti-glucagon (Catlog#14974382, IF/IHC, 1:200 dilution) from Thermo Fisher Scientific (Waltham, MA, United States), anti-p-H3S10 (Catlog#5176, IHC, 1:200 dilution) from Abcam (Cambridge, United Kingdom), and anti-Glut2 (Catlog#sc-9117, IF, 1:1000 dilution) from Santa Cruz Biotechnology (Dallas, TX, United States). Secondary antibodies used were: F(ab’)2-Goat anti-Mouse IgG-Alexa 594 (Catlog#A11020, IF, 1:800 dilution) and Goat anti-Rabbit IgG-Alexa 488 (Catlog#A31628, IF, 1:800 dilution) from Thermo Fisher Scientific (Waltham, MA, United States), anti-AKT (Catlog#9272, Western blot, 1:1000 dilution), anti-p-AKT (Ser473) (Catlog#9271, Western blot, 1:1000 dilution), and anti-Gapdh (Catlog#2218, Western blot, 1:1000 dilution) from Cell Signaling Technology (Danvers, MA, USA). Chemical reagents used were BSA (Catlog#A2153) and DAPI (Catlog#F6057) from Sigma-Aldrich (Oakville, ON, Canada).

For immunohistochemistry experiments, explants were fixed with 4% PFA for 10 mins at RT, followed by permeabilization with 0.1% NP40 for 30 mins at 37°C. Next, the cells were blocked with 1% BSA in PBS for 30 mins at 37°C, prior to incubation with the primary antibody for 1h at 37°C. Primary antibodies were prepared in blocking solution, and were used in the following dilutions: mouse anti-E-cadherin antibody (BD Biosciences) (1:200), mouse anti-Paxillin antibody (BD Biosciences) (1:200), and rabbit anti-Active Yap1 (Abcam)(1:200), anti-pH3(S10) (Abcam, 1:200), anti-Caspase3 (R&D Systems, 1:200). Following primary antibody incubation, the explants were washed 4X times in PBS for 10 mins and incubated in a secondary antibody cocktail for 30 mins at 37°C. Finally, cells were washed 3X times in PBS and stained with DAPI or Phalloidin for 20 mins at RT. Post antibody staining, imaging was performed using Andor/Olympus Spinning Disk Confocal microscope at BRC facility, Cornell University.For Proximity Ligation Assay (PLA), explants were fixed and permeabilized as described above.The primary antibodies used were: Mouse Anti-YAP1 (DSHB) (1:5) and Rabbit Anti-TEAD1 (Abcam) (1:200). Following primary antibody incubation, the remaining steps of PLA were performed using reagents from the Duolink PLA detection kit (Sigma Aldrich, DUO92101) according to the manufacturer’s protocol.