Phosphate buffered formaldehyde solution

Tissues were fixed in 4% phosphate-buffered formaldehyde solution (Fisher Scientific) for 24 hours. Fixed tissue sections were de-paraffinised and antigen retrieval performed in 0.01M sodium citrate buffer. Slides were blocked with goat serum, stained with mouse anti-E-cadherin (BD, 610181) and rabbit anti-C.rodentium antiserum followed by staining with secondary antibodies (AF555-conjugated goat-anti-rabbit and AF488-conjugated goat-anti-mouse from ThermoFisher). Slides were further stained with DAPI (Sigma) and mounted in Fluoromount-G (SouthernBiotech) and visualized using a Leica Confocal SP5-Invert microscope. For staining of eYFP, tissue sections were fixed in 4% paraformaldehyde at 4°C for 16 hours followed by incubation in 30% sucrose for 24 hours. Tissues were embedded in O.C.T. compound (VWR) followed by cryosectioning. Slides were blocked with rabbit serum and stained with AF488-conjugated rabbit-anti-GFP (ThermoFisher, A21311) and AF647-conjugated mouse-anti-E-cadherin (BD, 560062) and visualized using a Leica Confocal SP5-Invert microscope. Image analysis was performed in ImageJ.

Tissues from distal colon and caecum were fixed in 4% phosphate-buffered formaldehyde solution (Fisher Scientific) for 24 hours, cut and stained with hematoxylin and eosin. Slides were blinded and scored (0-15) for parameters of inflammation and tissue damage as described in28 (link).