Confocal Immunofluorescence of Colonic Tissue

Paraffin sections of colonic tissue (6 μm) were used for confocal analysis with antibodies raised against specific proteins of interest according to standard protocols (25 (link)). In brief, after slides were de-paraffinized and rehydrated, antigen retrieval was performed in citrate buffer (10 mM, pH 6.0, 30 min., 95°C). After blocking in 5% BSA (w/v) and normal goat serum, samples were incubated in primary antibody overnight (16 h, 4°C). Slides were washed extensively (3 x 5 mins) in TBS-tween20 and incubated in appropriately labeled secondary antibodies (Invitrogen) for 1 h at room temperature, washed, counterstained with DAPI in TBS-tritonX100 0.1% v/v, washed and mounted in Prolong gold (ThermoFisher, Waltham, MA). Staining using anti-mouse CDH1 (E-cadherin) and anti-mouse βIII Tubulin (Table S2) was revealed using a mouse-on-mouse kit according to manufacturer’s instructions (Vector laboratories, Burlingame, CA). Primary and secondary antibodies used are detailed in Table S2. Slides were imaged on a Leica SP8 STED 3X confocal microscope with a 40X 1.3NA objective or a 63X 1.4 NA objective. All areas larger than the field of view of the objective were acquired using a tiling approach, whereby adjacent images were acquired with a 10% overlap, and processed by Imaris Stitcher (Oxford Instruments United Kingdom).