2.4 Immunofluorescence staining
Immunofluorescence staining was conducted to confirm the TRPA1 expression on sensory nerves. Bladder tissues were fixed with 4% formalin for >24 h. Bladder tissues were embedded in paraffin and then cut in the transverse direction into sections of thickness 8μm. Sections were deparaffinized, rehydrated, and followed by antigen retrieval for 10 min. Then, sections were blocked with 5% albumin from goat serum in a 37 °C oven for 1 h, followed by incubation with mixed primary antibodies (TRPA1, 1:100, Alomone labs; Substance P, SANTA CRUZ, 1:100) overnight at 4 °C. Primary antibodies were visualized with Alexa Fluor 594-conjugated goat anti-mouse IgG (H+L; diluted 1:200 in phosphate-buffered saline, Elabscience Biotechnology Co., Ltd., Wuhan, China) or fluorescein-conjugated goat anti-rabbit IgG (H+L; diluted 1:50). Sections were analyzed by Confocal laser scanning microscope ZEISS Observer.Z1 (Carl Zeiss Microscopy GmbH, Baden Wurttemberg, Germany). Images were acquired with the ZEN 2.1 (blue version) (Carl Zeiss Microscopy GmbH, Baden-Wurttemberg, Germany). Control staining was performed with PBS rather than primary antibodies.