Citrate antigen retrieval buffer

Sections from the distal small intestine from each treatment group were fixed in 10% formalin and embedded in paraffin wax. A minimum of 10 sections from each of the 5 animals per group were analyzed by immunofluorescence microscopy. Representative images are shown. For immunostaining with Claudin-1 antibody, 8 μm tissue sections were deparaffinized with xylene and tissue was rehydrated through a series of graded alcohol and then processed for antigen retrieval using citrate antigen retrieval buffer (DAKO). Tissue sections were stained with polyclonal rabbit antibody against Claudin-1 (Invitrogen) in PBS with 1% bovine serum albumin (BSA) overnight at 4°C. For immunostaining with anti-ZO-1 antibody, intestinal tissue was frozen in TFM tissue freezing media, and later 5 μm tissue sections were used. Sections were fixed with 1% paraformaldehyde for 10 min at room temperature and incubated with anti-ZO-1 antibody (Invitrogen). After washing, sections were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (Invitrogen); for F-actin staining, sections were incubated with Alexa 555 conjugated phalloidin (Invitrogen) for 1 h at room temperature. Sections were mounted under coverslip using ProLong Gold antifade reagent with DAPI (Invitrogen). Stained sections were imaged using a fluorescence microscope (Leica Microsystems, Germany).

Excised ileal section was fixed in 10% neutral buffer formalin for 24 hours and embedded in paraffin. 8 µm section was mounted on microscopy glass slide and stained with hematoxylin and eosin (H&E) for histological evaluation. H&E-stained slides were randomized and coded for histological evaluation in a blinded fashion. For immunofluorescence staining with tight junction protein, fixed ileal sections were deparaffinized using xylene and passed through series of graded alcohol for rehydration. Heat antigen retrieval was performed using citrate antigen retrieval buffer (DAKO) and tissue section was blocked with 5% BSA. Ileal tissue sections were stained with anti-Claudin-1 antibody (Invitrogen, catalog no. 51–9000) at a dilution of 1:100 in PBS + 1%BSA overnight at 4°C. Anti-rabbit Alexa 488 conjugated secondary antibody (Invitrogen, catalog no. A-11008) was added at a dilution of 1:800 for 1 hour at room temperature. Ileal tissue section was mounted under coverslip using ProLong Gold antifade mounting media with DAPI (Invitrogen, catalog no. P36935) and visualized using fluorescence microscope (Leica Microsystems, Germany).