Sp8 sted 3x confocal microscope

To stain the recorded cells, biocytin (3 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) was added to the intracellular solution. Cells were held in whole-cell configuration for more than 15 min to allow biocytin diffusion into their cytosol. Following electrophysiological recordings, slices were (i) fixed in 4% paraformaldehyde for 1 h, (ii) rinsed with phosphate-buffered saline (PBS; Dulbecco’s, Sigma), (iii) rinsed alternately (16 washes of 10 min each) with quenching buffer (QB; glycine 0.1 M in PBS 120 mM) and blocking buffer (BB; BSA 1% and Triton X 0.3% in PBS 120 mM), (iv) incubated overnight with 5 µg/mL Alexa Fluor 568-conjugated streptavidin (Thermo Fisher Scientific, Waltham, MA, USA), (v) rinsed alternately (16 washes of 10 min each) with QB and BB, (vi) incubated for 30 min at 4 °C with 10 µg/mL DAPI (4′,6-diamidino-2-phenylindole; Molecular Probes), (vii) rinsed with PBS, (viii) mounted on microscope slides using ProLong TM glass antifade mountant (Thermo Fisher Scientific, Waltham, MA, USA), and (ix) stored in the dark at 4 °C until acquired via confocal microscopy (Leica SP8 STED 3x Confocal Microscope, Leica Microsystems, Wetzlar, Germany, and LAS X Life Science Software, version 3.7.4.23463).

To determine where the Cj0371 carries out its function in C. jejuni, we used GFP tagged Cj0371 to investigate its subcellular localization by fluorescence microscopy. First, we constructed C. jejuni Δcj0371 expressing a functional green fluorescent protein of Cj0371. Using primers cj0371-F3 and cj0371-R3 (Table 2), we amplified cj0371 without its termination codon from C. jejuni 11168, and cloned it into pMD-19T to generate pMD-19T-cj0371. BamH I was used to digest pMD-19T-cj0371 and pUOA18-PmetK, then the cj0371 fragment was cloned into pUOA18-PmetK to generate pUOA18-PmetK-cj0371, following by cloning the PmetK-cj0371 fragment into pRY107-egfp. The recombinant plasmids pRY107-PmetK-cj0371-egfp were then transformed into E. coli DH5ɑ and subjected to restriction analysis to confirm that they carried the desired sequence. The plasmids were introduced into C. jejuni Δcj0371 by biparental conjugation. Transconjugants were selected and confirmed as described above. The resulting strains were grown in MH liquid medium to OD₆₀₀ of 0.4 and washed with PBS once. Ten microliters of the culture volume was loaded on slides, then spotted with 10 μl 4% paraformaldehyde in the bacterial suspension and observed on a Leica SP8 STED 3X confocal microscope.

Slides with colonic tissue section were also used for confocal analysis with antibodies raised against specific proteins of interest according to standard protocols. 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 and normal donkey serum, samples were incubated in primary antibody overnight (16 h 4°C). Primary and secondary antibodies used are detailed in Table 1. 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 (Invitrogen). Staining using anti-mouse CDH1 (E-cadherin) was revealed using a mouse on mouse kit according to manufacturer’s instructions (Vector laboratories, Burlingame, CA). Slides were imaged on a Leica SP8 STED 3X confocal microscope with a 63X 1.4 NA objective. 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.

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).