Immunohistochemical Analysis of TG2, TRPA1, and TRPV1 in EB

Paraffin-embedded lung tissue sections were deparaffinized and the antigen were repaired using microwave oven heating. Bronchial brushes of EB or control patients were donated with ethical approval from the Ethics Committee of the 1st affiliated Hospital of Guangzhou Medical University (No. 2017-27). All methods were carried out in accordance with relevant guidelines and regulations, and informed consent was obtained from all donors or, if donors are under 18, from a parent and/or legal guardian.TG2 expression level and localization in the lung tissue from EB or control mice with or without CTM treatment, or bronchial brushings from EB or control patients were detected through immunohistochemical assay (IHC). Lung tissues were stained overnight with anti-TG2 (CST, 3557 s) at 4 °C, followed by HRP-conjugated secondary anti-rabbit antibody. Anti-TRPA1(NOVUS, NB110-40763) and Anti-TRPV1(Abcam, ab31895) were incubated overnight at 4 °C on the mice lung sections with or without CTM treatment, subsequently with goat anti-mouse or rabbit cross-adsorbed secondary antibody for 30–40 min at room temperature. The images were observed and captured with an optical microscope (Olympus, Japan) for IHC and fluorescence microscope (ZEISS, Germany) for IF. The semi-quantitative IHC assay was also determined by Image J software and then modified H-score^{47 (link)}.