Mouse anti acetylated α tubulin

AOs were differentiated using described protocols. On the day of the analysis, organoids were harvested and washed in 10 ml DMEM+P/S. Organoids were dissociated into single cells using TrypLE (Gibco) supplemented with Y‐27632 at 37°C for 30 min. During the single‐cell dissociation, the organoid suspension was vigorously pipetted every 5 min to keep the solution homogenous. Cells were fixed in 4% paraformaldehyde for 2 h at RT. The organoids were permeabilized for 30 min in 0.5% Triton X‐100 (Sigma) and blocked for 30 min in 2% normal goat serum in PBS. Organoids were incubated with primary antibody (mouse‐anti‐acetylated‐α‐tubulin; Santa Cruz; sc‐23950) for 1 h at RT, washed three times with PBS, incubated with secondary antibodies (goat‐anti‐mouse IgG‐Alexa488; Invitrogen) for 1 h at RT and washed two times with PBS. Cells were strained over 35 µm mesh into Falcon® 5 ml Round Bottom Polystyrene Test Tube (Corning; 352235). FACS analysis was performed within 30 min on BD LSR Fortessa X20 4 laser FACS machine. Data analysis was performed using FlowJo (v10.7.2).

Western blot protocols have been previously published [23 (link)]. Immunoprobing was performed using the following antibodies: mouse anti-acetylated α-tubulin (Santa Cruz Biotechnologies, Dallas, TX, USA), rabbit anti-actin (Sigma Aldrich), rabbit anti-Notch3 (Abcam, Cambridge, UK), rabbit anti-Notch1 (Cell Signaling), mouse anti-c-Myb (Thermo Fisher Scientific), rabbit anti-HDAC6 (Santa Cruz Biotechnologies), rabbit anti-Histone H3 (Cell Signaling or Abcam), rabbit anti-acetyl-Histone H3 (Lys 9) (Cell Signaling), mouse anti-LAMP2 (Novus Biologicals, Littleton, CO, USA), mouse anti-Pan-cadherin (Santa Cruz Biotechnologies), mouse anti-GAPDH (Santa Cruz Biotechnologies), rabbit anti-VDAC (Cell Signaling) followed by incubation with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary Ab (Perkin Elmer, Waltham, MA, USA). Antigens were identified using Western Lightning plus ECL (Perkin Elmer) or ECL Select Western Blotting detection (GE Health Care) reagents and detected by UVITec Alliance LD2 (UVITec Cambridge, UK) imaging system.

Immunohistochemistry was performed as previously described (Sulkowski et al., 2011 (link)). Primary antibodies used were rabbit anti-PP2CB (used at 1:50 dilution) (Biorbyt); chicken anti-GFP (used at 1:1000 dilution) (Abcam); mouse anti-Cut (used at 1:100 dilution) (DSHB); mouse anti-Futsch (22C10) (used at 1:100 dilution) (Developmental Studies Hybridoma Bank); mouse anti-acetylated α-tubulin (used at 1:100) (Santa Cruz); rabbit anti-pS172 β-tubulin (used at 1:100 dilution) (ab78286, Abcam); rabbit anti-FoxO (used at 1:100 dilution) (ab195977, Abcam). Donkey anti-chicken 488 (1:1000) (Jackson Immunoresearch), donkey anti-rabbit 555 (1:200) (Life Technologies), donkey anti-mouse (1:200) (Life Technologies), and Alexa-Fluor goat anti-horseradish peroxidase (HRP) 647 (1:200) were used as secondary antibodies.