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.