Flouromount aqueous mounting medium

Brain sections were washed 3 times for 10 min in TBST and then blocked in TBS++ (TBS, 3% donkey serum – 130787, Jackson ImmunoResearch, 0.25% triton X-100 – T8787, Sigma Aldrich) for 1.5 h, followed by 72 h incubation on rocking platform at 4 ˚C in primary antibodies (see antibodies listed below). For secondary staining, brain sections washed 3 times for 10 min in TBST, followed by 1.5 h incubation in secondary antibody mix. Following secondary antibody incubation, there were 4 10 min washes in TBST; the second wash of which contained Hoechst (1:2000) with TBST. Brain sections were mounted on Superfrost™ microscope slides (12–550-15, Fisher Scientific) with Flouromount™ Aqueous Mounting Medium (Sigma-Aldrich, F4680). Slides were stored at 4˚C.

The organoid immunofluorescent staining was performed on 8 well chamber slides (Nunc, Rochester, NY) as fully described in the Supplementary Materials and Methods. Briefly, the organoids were fixed with warm 4% PFA on chamber slides for one hour at RT and then permeabilized with 1% Triton-X100. The samples were blocked with a solution of 3% goat serum/1% BSA/0.2% Triton X-100 for 1 hour. Primary antibody staining for the organoids was done overnight. Organoids were washed and incubated for 2 hours with the appropriate secondary antibody conjugated to Alexa Flour 488 (green). Organoids were then washed and double stained for DAPI and mounted using Flouromount aqueous mounting medium (Sigma Aldrich). Organoids were imaged using a Zeiss LSM 700 confocal microscope (Zeiss, Oberkochen, Germany). The mouse colon tissue immunofluorescent and immunoperoxidase staining was performed on paraffin-embedded formalin-fixed proximal colon tissue samples and is fully described in Supplementary Materials and Methods. Specific antibodies used for staining are detailed in Supplementary Table 2. All organoid and tissue staining data is from n = 4 mice/group with 7–10 images of stained organoids (or tissue) to determine relative expression of each marker and to pick the representative images. Staining was evaluated by two observers blinded to treatment group for all staining data.