Sc 15334

Immunofluorescence was performed as previously described (Li et al. 2018 (link)). Briefly, organoids were fixed in 4% paraformaldehyde for 1 h at room temperature. Organoids were washed by PBS for 3 times and permeabilized by 0.5% Triton X-100 for 1 h at room temperature. Then, samples were blocked with PBT solution (3% BSA and 0.01% Triton X-100 in PBS) for 2 h at room temperature and incubated with primary antibodies overnight at 4 °C. The fluorescein-labeled secondary antibodies (Life Technologies, 1:300) and 4′, 6-diamidino-2-phenylindole (DAPI) were added for 1 h at room temperature next day. The following antibodies were used for immunofluorescence: rabbit anti-Fabp1 (Abcam, ab171739, 1:300); mouse anti-E-cadherin (B&D, 610182, 1:300); rabbit anti-Muc2 (Santa Cruze, sc-15334, 1:300); rabbit anti-ChgA (Abcam, ab15160, 1:300). The images were acquired from Olympus FV3000 Laser Scanning Microscope.

For immunofluorescence staining, cells in both channels were first gently washed with PBS, then fixed with PFA (4%; Electron Microscopy Sciences, 157–4) for 20 min and subsequently washed with additional PBS. Cells were then permeabilized with 0.25% Triton X-100 (0.25%; Sigma, T8787) for 20 min and incubated with blocking buffer containing 1% BSA (Sigma, A4503) and 10% donkey serum (Sigma, D9663) for 30 min at room temperature. Cells were then incubated with antibodies directed against ZO-1 (Life Technologies, 33-9100, dilution 1:200), PECAM-1/CD31 (eBioscience, BMS137, dilution 1:100), VE-cadherin/CD144 (BD Biosciences, 555661, dilution 1:200), Villin (Life Technologies, PA5-29078, dilution 1:100), 53BP1 (Abcam, ab36823, dilution 1:100), MUC2 (Santa Cruz Biotechnology, sc-15334, dilution 1:100), or HIF-1α (Abcam, ab16066, dilution 1:100) overnight at 4 ^oC, followed by 6 × 5 min PBS washes. Secondary antibodies (Life Technologies) were then introduced in the channels for 1 h at room temperature and washed three times with PBS. Cells were co-stained with DAPI (Invitrogen, D1306). Microscopy was performed with a laser scanning confocal microscopy (Leica SP5 X MP DMI-6000 or Zeiss TIRF/LSM 710). Quantification of the immunofluorescence images was performed using ImageJ software based on the mean fluorescence intensity on a per cell basis.

Tissue was excised and fixed for 48 h in 4% PFA in PBS at 4°C, after which it was transferred to 20% sucrose solution. After cryosectioning, antigen retrieval was accomplished by incubating the slides in 1% SDS for 5 min. Blocking was performed with 10% donkey serum. Following washing, primary antibodies were added and incubated overnight at 4°C. The following primary antibodies were used: rabbit FITC-anti-Lyz (1:400, Dako, F037201), rabbit anti-Muc2 (1:50, Santa Cruz Biotechnology, sc-15334), rabbit anti-ChgA (1:100, Abcam, ab15160) and rabbit anti-Dclk1 antibody (1:1000, Abcam, ab31704). Secondary detection was carried out using Alexa Fluor 488 donkey anti-rabbit secondary antibody (1:500, Thermo Fisher Scientific, A-21206) and DAPI (10 μg/mL). Fluorescent imaging was carried out using a TCS SP5 confocal microscope (Leica). Image analysis was carried out using iMaris software.