A0099

After time-lapse imaging, organoids were fixed with 4% formaldehyde (Sigma) at room temperature for 30 min. Next, they were permeabilized with 0.2% Triton-X-100 (Sigma) for 1 hr at 4°C and blocked with 5% skim milk in Tris-Buffered Saline (TBS) at room temperature for 1 hr. Subsequently, organoids were incubated in blocking buffer containing primary antibody (rabbit anti-lysozyme 1:800, Dako #A0099) overnight at 4°C and then incubated with secondary antibody (anti-rabbit conjugated to Alexa Fluor405 1:1,000, Abcam #ab175649) at room temperature for 1 hr. Afterward, they were incubated with wheat germ agglutinin (WGA) conjugated to CF488 A (5 μg/ml Biotium) at room temperature for 2 hr, followed by incubation with RedDot1 Far-Red Nuclear Stain (1:200, Biotium) at room temperature for 20 min. Finally, organoids were overlaid with mounting medium (Electron Microscopy Sciences). The procedure was performed in the same imaging chambers used for time-lapse imaging in order to maintain organoids in the same position. Imaging was performed with the same microscope as previously described. Note that WGA stains both Paneth and Goblet cells, but the lysozyme staining allowed the unequivocal distinction between them.

Immunostaining for lysosome, Sucrase-Isomaltase, Ki-67, mMECA-32, and ChromograninA was performed on frozen sections using rabbit anti-lysozyme (A0099, DAKO, 1:1000), mouse anti-sucrase-isomaltase (sc-393470, clone: C-8, Santa Cruz, 1:50), rabbit anti-ki-67 (ab16667, clone: SP6, abcam, 1:500), rat anti-MECA-32 (NB100-77668, clone: MECA-32, NOVUS Biologicals, 1:50), and rabbit anti-ChoromograninA (ab15160, Abcam, 1:400) antibodies, respectively. Immunostaining for GFP to detect Lgr5 expression was performed on frozen sections or paraffin-embedded sections using rabbit anti-GFP (2956, clone: D5.1, Cell Signaling, 1:100) antibody. For DAB staining, slides were then incubated with amino acid polymer conjugated with peroxidase and Fab’ (Histofine simple stain rabbit MAX-PO (R), 414341, Nichirei Bioscience Inc.). Slides were visualized using metal-enhanced 3,3′- diaminobenzidine (DAB) and counterstained with hematoxylin. Images were acquired using an OLYMPUS BX41 microscope (OLYMPUS Corporation). When co-staining of Lys and GFP or co-staining with rainbow colors were performed, Alexa488-, Alexa594-, and Alexa750-conjugated secondary antibodies (1:200, Invitrogen) were used, followed by nuclei-staining using Hoechst33342 (Thermo Fisher Scientific, H3570). Fluorescent images were taken with an OLYMPUS BX63 microscope (Olympus Corporation, Tokyo, Japan).