Confocal Imaging 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, Carlsbad CA) 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 (S2 Table) 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 S2 Table. 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).