2.7. Immunostaining and Confocal Imaging
Organoids were fixed for 1 hour at room temperature with 4% PFA. Samples were then blocked for 1 hour at room temperature using goat blocking solution (5% Normal Goat Serum with 0.1% w/v Sodium Azide) with 0.1% Triton-X. Primary antibodies (anti-Connexin 43 N-terminal antibody purchased from Sigma-Aldrich), were diluted in goat blocking solution and samples are incubated overnight at 4°C. Samples were then washed with PBS 3 times for 5 minutes each. Secondary antibodies were also diluted in goat blocking solution and incubated with samples overnight at 4°C. Samples were then washed with PBS 3 times for 5 minutes and stained with DAPI (NucBlue, Invitrogen) and Allexa-647 conjugated Phalloidin (Invitrogen) per the manufacturer instruction. Imaging was conducted using Nikon A1R HD Confocal Microscope.