Permount sp15 100

H&E staining was conducted for the evaluation of histological changes in colon tissue according to the method described earlier [44 (link),45 (link)]. Paraffin slides were deparaffinized and rehydrated in xylene and ethanol. These slides were treated in Meyer’s hematoxylin (S3309, DAKO, Glostrup, Denmark) for 30 s and washed with water. These slides were incubated with eosin (318906, Sigma-Aldrich, St. Louis, MO, USA) for 10 s and washed with water. These slides were air-dried at room temperature and then sequentially treated in ethanol and xylene. Coverslips were covered onto the slides and mounted by Permount^® (SP15-100, Thermo Fisher Scientific, Waltham, MA, USA). Ulcerative colonic damage was evaluated with the Wallace microscopic score (Table 1) [46 (link),47 (link)].

To evaluate epidermal thickening, the ear and dorsal skin of each mouse was obtained on day 21 and fixed in 10% neutral buffered formalin. Tissues were embedded in paraffin and sliced into 5-μm-thick sections. Sections were then transferred to probe-on-plus slides (Thermo Scientific, Carlsbad, CA, USA), and deparaffinized skin sections were stained with H&E. Mast cells in the skin were stained with toluidine blue. Some sections were stained for immunohistochemical markers using anti-filaggrin (1:1000; ab24584; Abcam), anti-involucrin (1:1000; ab68; Abcam), anti-loricrin (1:1000, ab85679, Abcam), anti-occludin (1:1000; 33-1500; Invitrogen), anti-PAR-2 (1:100; sc-8205, Santa Cruz Biotechnology), and anti-TSLP (1:500; NB110-55234; Novus Biologicals) antibodies. The staining procedure was performed with an Ultravision Quanto Detection System (TL-060-QHD; Thermo Scientific). The slices were washed with PBS, dehydrated and mounted in Permount (SP15-100, Thermo Scientific). All stained sections were then examined by light microscopy (DM750, Leica, Wetzlar, Germany) to assess histological changes. Morphometric analysis (immunostained area of the epidermis to each analyzed protein in relation to total area of the epidermis) was performed using ImageJ 1.51 software (National Institutes of Health, Bethesda, MD, USA). All histological examinations were analyzed in 3 sections/animal slices.

Each tissue slide was prepared as described previously [69 (link)]. Staining was carried out via H&E and toluidine blue, followed by immunohistochemical staining with anti-filaggrin (Abcam, Cambridge, UK), anti-loricrin (Abcam), and anti-TRPA1 (GTX54765; GeneTex, Irvine, CA, USA) antibodies. Staining was performed using an Ultravision Quanto Detection System (TL-060-QHD; Thermo Fisher Scientific, Waltham, MA, USA). After staining, tissues were dehydrated and sealed in Permount (SP15-100; Thermo Fisher Scientific), and observed via optical microscopy (DM750, Leica, Wetzlar, Germany). For fluorescence images, the sections were incubated with primary antibody with anti-TRPA1 (GTX54765; GeneTex, Irvine, CA, USA) antibody at 4 °C for 16 h, followed by incubation with secondary antibodies against FITC-conjugated goat-anti-rabbit IgG (1:1000, sc-2012, Santa Cruz Biotechnology) at 21 °C for 1 h. Immunostained tissues were mounted with a medium containing Fluoroshield™ with DAPI (ImmunoBioScience, Mukilteo, WA, USA). Fluorescence images were acquired by using a confocal microscope (LSM700; Zeiss, Jena, Germany).