Fluoview fv1000 confocal microscope 60x oil lens

Pig gallbladder organoids were mechanically dissociated from the Matrigel, washed, and fixed in 4% PFA for 15 minutes at 4° C. Organoids were kept in suspension at 4° C while they were permeabilized in 0.03% Triton for 1 hour, and blocked in Superblock (Thermo-Fisher) with 4% normal goat serum for 48 hours. Organoids were then incubated with primary antibodies overnight: mouse anti-CFTR (clone 769) (1:100 dilution, University of North Carolina – Chapel Hill and the Cystic Fibrosis Foundation Therapeutics) and rabbit anti-Na⁺/K⁺-ATPase (clone EP1845Y) (1:100 dilution, Abcam). Organoids were then washed with PBS and incubated with goat anti-mouse and goat anti-rabbit secondary antibodies conjugated to Alexa-Fluor 488 and Alexa-Fluor 568 (1:1000; Molecular Probes/Invitrogen), respectively. Organoids were washed again with PBS. Organoids were then incubated with Alexa-Fluor 633 conjugated phalloidin (1:40; Molecular Probes/Invitrogen) for 1 hour at room temperature. Organoids were then washed in PBS, mounted with Vectashield plus DAPI (Vector Labs), coverslipped, and visualized with an Olympus Fluoview FV1000 confocal microscope 60x oil lens.