P0425 72ea

Isolated cells (2,000–3,000 in 5–10 µl of IMDM) were pipetted onto poly-L-lysine coated slides (P0425-72EA; Sigma-Aldrich), settled down for 15 min at RT, fixed with 4% paraformaldehyde for 10 min at RT, then washed three times with PBS, and permeabilized/blocked for 1 h at RT with 0.1% Tween-20 in 10% FBS (Corning) in PBS, which was then used as antibody incubation buffer for all the subsequent steps. Cells were incubated overnight at 4°C with a mouse monoclonal anti-KDEL (ab12223; Abcam) or a rabbit anti-mouse β-catenin (9582S; Cell Signaling) primary antibody, washed three times with PBS, and incubated for 1 h at RT with a goat anti-mouse IgG A488 (A11029; Invitrogen) or a donkey anti-rabbit-A555 (A31572; Invitrogen) secondary antibody. Cells were then washed three times with PBS, stained with 1 μg/ml DAPI (32670; Sigma-Aldrich) for 10 min at RT, washed three times with PBS, and finally slides were mounted with VectaShield (H-1000; Vector Laboratories). Cells were imaged on a Nikon Ti Eclipse inverted confocal microscope with 60× objective using Nikon NIS Elements (v3) for data collection, and images were processed using Fiji (https://fiji.sc). Cells were imaged on an Olympus epifluorescence microscope with 60× objective for manual scoring of nuclear β-catenin staining. At least 100 cells per condition were randomly captured for quantification.