Ab24584

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.

The dorsal skin was excised from the mice after sacrifice by isoflurane. The skin species were immersed in a 10% buffered formaldehyde using ethanol, embedded in paraffin wax, and sliced at a thickness of 3 mm. The samples were stained by hemoxylin and eosin (H&E) and imaged under light microscopy (IX81, Olympus, Tokyo, Japan). Immunohistochemistry was performed using the Polink-2 HRP Plus Rabbit DAB Detection System (Golden Bridge International, Inc.) according to manufacturer’s recommendations, the primary anti-COX2 (1:500, ab15191, abcam) and anti-filaggrin (1:500, ab24584, abcam) antibodies were incubated with the skin specimens at room temperature for 1 h. The skin sections stained with COX2 and filaggrin were visualized by light microscopy.