E0432

At the conclusion of the experiment, implanted rats were euthanized and the TNBS-inflamed segment of small intestine tissue dissected and processed as previously described (Payne et al., 2018c (link)). In brief, a 2 cm segment of control tissue was removed spanning 5–7 cm oral to where the ligation limiting the inflamed site had been. As TNBS-induced inflammation is patchy, the 8 cm length of inflamed intestine was divided equally into four 2 cm long segments, and cut longitudinally along the mesenteric border and pinned out onto balsa boards (mucosa side up). One half was placed in fixative (2% formaldehyde plus 0.2% picric acid in 0.1 M sodium phosphate buffer, pH 7.4) overnight, embedded in paraffin, sectioned (5 μm) and stained with hematoxylin and eosin (H&E) (Pontell et al., 2009 (link)) or immunohistochemically stained with anti-CD3, a cytotoxic T cell marker (1:200; Cytomation, Dako E0432) (Payne et al., 2018c (link)). The other half of the tissue was processed for frozen sections (14 μm) and myeloperoxidase (MPO) staining (Payne et al., 2018c (link)). All sections were mounted with DPX.

For immunostaining on mouse brain sections, 5 µm sections were prepared from paraffin-embedded samples. Sections were dewaxed using Varistain (Thermo Fisher Scientific), followed by an antigen retrieval step using citrate buffer (Vector; H-3300) and wash steps with PBS-T (PBS supplemented with 0.5% Triton X-100). Next, endogenous peroxidase activity was blocked with 3% H₂O₂ in methanol for 10 min. Samples were blocked with bovine serum albumin (BSA) and 5% normal goat serum in PBS-T (0.5% (w/v) BSA and 0.02% (v/v) Triton X-100 in PBS) for 30 min at RT, followed by overnight incubation at 4 °C with primary antibody against GFAP (1:1000; Agilent; Z033429-2) or IBA1 (1:200; Wako Chemicals, 019-19741). After ON incubation, slides were washed with PBS-T and incubated with goat anti-rabbit biotinylated antibody (1:500; Dako, E0432). Visualization was done using ABC (Vector; PK6100) and DAB. Next, slides were counterstained with hematoxylin, dehydrated to xylene and mounted with Entellan. Image acquisition was performed using a slide scanner (Zeiss, Axio Scan) and analyzed using the Zen software (Carl Zeiss Microscopy GmbH, Jena, Germany, 2012).

Human dermal and lung fibroblasts were treated for 15, 30 and 45 min. with (i) ACHP, (ii) TGFβ1, or (iii) both. After treatment, cells were washed with PBS and fixed either with methanol/acetone (1:1) (Smad2/3) for 5 min. or with 0.5% para‐formaldehyde (RelA) for 15 min. Subsequently, cells were washed with PBS and incubated either with polyclonal goat‐anti‐human to Smad2/3 (AF3797; R&D, Abingdon, UK) diluted in a concentration of 15 μg/ml, or with polyclonal rabbit anti‐human to RelA (ab16502; Abcam) at 1:50 dilution in PBS containing 2% BSA for 3 hrs at 4°C. After washing with PBS, cells were incubated with biotinylated secondary antibody rabbit anti‐goat to detect Smad2/3 (6160‐08; SouthernBiotech) and goat‐anti‐rabbit to detect RelA (E0432; Dako) diluted in PBS (1:100) containing 2% BSA for 30 min. at RT. The cells were washed again and incubated for 30 min. with streptavidin‐CY3 (1:100) in PBS containing 1% BSA and DAPI (1:10,000). After washing with PBS, stained wells were mounted with Citifluor and the translocation of RelA and Smad2/3 was visualized by using confocal laser scanning microscopy (Leica TCS SP8; Leica Microsystems GmBH, Wetzlar, Germany).

The slides were deparaffinized in xylene and then rehydrated in a graded series of ethanol, followed by antigen retrieval in a microwave. To block the activity of endogenous peroxidase, the slides were incubated with 10% rabbit serum and 3% hydrogen peroxide for 10 min at room temperature. Then, the slides were incubated with a PPAR gamma antibody (ab59256, Abcam, Cambridge, UK; 1:500) overnight at 4℃. Then, washing twice in Tris-buffered saline and incubating the slides with goat anti-rabbit polymers (E0432, Dako, Glostrup, Denmark) at rt for. Using a standard streptavidin-biotin complex (Sigma, MO, USA) for the final test and using Olympus BH2 microscope (Olympus, Tokyo, Japan) to evaluate the results.

For immunostaining on mouse brain sections, 5‐μm sections were prepared from paraffin‐embedded samples. After dewaxing, samples were treated with citrate buffer (Dako; S2031), followed by washing in PBS. Endogenous peroxidase activity was blocked with 3% H₂O₂ in methanol for 10 min, and samples were blocked with 5% goat serum in antibody diluent (Dako; S2022) for 30 min at RT, followed by overnight incubation at 4°C with primary antibody against claudin‐1 (CLDN1, Invitrogen, 51‐9000, 1:1,000) or IBA1 (Wako Chemicals, 019‐19741, 1:500). The next day, slides were washed with PBS and incubated with secondary antibody coupled to HRP or biotin, respectively (Dako; EK4003 and E0432). An amplification step was performed using tyramide (TSA kit, Perkin Elmer) for CLDN1‐staining, and visualization was done using ABC (Vector) and DAB. Slides were counterstained with hematoxylin, dehydrated, and mounted with Entalan. Slides were scanned using a slide scanner (ZEISS, Axio Scan) and analyzed with the Zen software (Carl Zeiss Microscopy GmbH, 2012). Histological quantification of the plaques in the whole brain was done with the Fiji software (University of Wisconsin‐Madison) by color thresholding the brown color, using the exact same parameters for all genotypes.