Sp3 confocal microscope

Mammary organoids were fixed with 4% paraformaldehyde at room temperature for 10 minutes. The organoids were washed twice with PBS, incubated with methanol for 2 minutes, followed by a 2 minutes incubation in PBS with 1% Triton-X. The organoids were permeabilized by incubating with penetration buffer (0.2% Triton-X, 0.3M glycine, 20% DMSO in PBS) for 15 minutes. Samples were then blocked with blocking buffer (5% mouse serum, 5% rabbit serum, 5% goat serum, 2% bovine serum albumin, 0.5% Triton X, 10% DMSO in PBS). MCL-1 (1:100, Santa Cruz, sc-20679) primary antibody was diluted in antibody buffer (50% blocking buffer, 0.2% Tween-20) and samples were incubated for 1 hour at room temperature on a shaker. Secondary antibody incubation with goat anti-mouse Alexa Fluor 488 (ThermoFisher A-11001) conjugated antibodies at a dilution of 1:200 was performed in the dark at room temperature for 1 hour. Samples were then washed five times before mounting on a slide using PBS:glycerol (1:1). Images were captured using a Leica SP3 confocal microscope. Images were pseudo-colored using ImageJ.