Rabbit anti pdx 1

Proliferative beta cells were detected in the pancreatic sections by double immunofluorescence staining with guinea pig anti-insulin (1:1000; DAKO), rabbit anti-Ki67 (1:200; Thermo Fisher, Burlington, ON, Canada) antibodies, and relevant secondary antibodies (1:1000; Abcam). Regenerative beta cells were detected by double immunofluorescence staining with guinea pig anti-insulin (1:1000; DAKO), rabbit anti-PDX-1 (1:400; Cell Signaling Technology, Danvers, MA, United States) antibodies, and relevant secondary antibodies (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA, United States). Apoptotic beta cells were also identified in pancreatic sections with insulin and terminal deoxynucleotidyl transferase dUTP nick end labeling (Tunel) labeling (TMR red, Roche, Mississauga, ON, Canada) (Wang and Brubaker, 2002 (link); Robertson et al., 2008 (link)). Results are expressed as the percentage of Ki67⁺, PDX-1⁺, or Tunel⁺ beta cells. All immunofluorescent images were captured by an Olympus upright BX50 fluorescence microscope (Olympus, Richmond Hill, ON, Canada) at ×40 magnification.

Media was aspirated away from Matrigel domes. Matrigel domes were washed with 500 μL room temperature PBS. Wells were fixed with 4% paraformaldehyde in PBS for 30 mins at room temperature. Wells were rinsed 3 × 10 min with 500 μL 100 mM glycine in Tris pH 7.4. Cells were permeabilized with 500 μL 0.5% Triton X-100 in PBS at room temperature for 5 min. Wells were washed in IF wash buffer (0.1% BSA, 0.2% Triton X-100, 0.05% Tween 20) 3 × 10 min. Wells were incubated with 500 μL of IF wash buffer with 1% BSA to block for 1 h at room temperature. Block buffer was aspirated and wells were incubated with primary antibody (mouse anti-αSMA, AbCam; mouse anti-Maspin, BD-Pharmingen; Rabbit anti-PDX1, Cell Signaling Technology; mouse anti-CK19, AbCam) 1:200 in 500 μL block buffer for 1 h at room temperature. Wells were washed 3 × 20 min in IF wash buffer. Wells were incubated with secondary antibody (Anti-Mouse Alexafluor Texas Red, ThermoFisher or Anti-Rabbit Alexaflour 488) 1:500 in 500 μL block buffer for 1 h at room temperature. Wells were washed 3 × 20 min. Wash was removed thoroughly and 150 μL of SlowFade anti-fade mountant with Dapi (ThermoFisher). Organoids were observed and micrographed on a Zeiss inverted microscope with fluorescence and Zen analysis software (Zeiss).