Ab31895

DSM samples were fixed in 4% formaldehyde and cryoprotected in 30% sucrose. The bladders were frozen in OCT compound and then cut into 5 μm transversal sections. The specimens were blocked in PB containing 5% bovine serum albumin, 10% normal goat serum, and 0.3% Triton X-100 for 2 h at room temperature. Next, the sections were allowed to incubate with the primary antibodies rabbit anti-TRPA1 (ab58844, 1 : 100, Abcam, Cambridge, UK) [18 (link)] or rabbit anti-TRPV1 (ab31895, 1 : 200, Abcam, Cambridge, UK) [19 (link)] and mouse anti-PGP 9.5 (ab8189, 1 : 100, Abcam, Cambridge, UK) for 48 h. After washing, the sections were incubated with secondary antibodies Alexa Fluor 488 goat anti-mouse (A-11029, 1 : 200, Invitrogen, Madrid, Spain) and Alexa Fluor 594 goat anti-rabbit (A-11037, 1 : 200, Invitrogen, Madrid, Spain). The cell nuclei were stained using DAPI (P36935, Molecular Probes, Eugene, USA). The intensity of fluorescence was evaluated using free software, ImageJ (USA).

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)}.

For double-immunofluorescence of TRPV1 and PSD95 (a marker for post synaptic mechanisms), free-floating sections containing PFC were washed in PBS and then preincubated in PBS containing 5% normal goat serum for 3–4 h at RT. After preincubation, sections were incubated with polyclonal rabbit anti-TRPV1 antibody (ab31895, Abcam, dilution: 1:500), and mouse monoclonal antibody anti-PSD95 (ab13552; Abcam; 1:500) for 24 h at 4°C, after which they were rinsed 3–5 times in PBS, the sections were then incubated for 1–2 h at RT in goat anti-rabbit IgG Alexa Fluor 488 (ab150077; Invitrogen; 1:1500) and goat anti mouse IgG Alexa Fluor 647 (ab150115; Abcam; 1:1500) dissolved in blocking solution. Following the final rinsing, the slices were wet mounted onto subbed slides and subsequently dried and cover-slipped with Dako Faramount Aqueous Mounting Medium (Dako; S3025). For fluorescence imaging, tissues were visualized under ab epifluorescence IX81 microscope (Olympus), and for confocal imaging a 700-AX10 laser scanning microscope (Carl Zeiss) was used.