Ls c180179

The distal colon tissues and DRGs were fixed with 4% paraformaldehyde for 24 h, and cross-sections of the colonic tissues were obtained after being embedded in paraffin. The sections were stained with diaminobenzidine for 15 s. All sections were blocked with 5% goat serum and incubated with the primary antibody at 4°C overnight. The sections were then washed three times in phosphate-buffered saline (PBS) and incubated at room temperature with the secondary antibody for 1 h. For immunofluorescence analysis, the nuclei were counterstained with 4’,6‐diamino‐2‐phenylindole (DAPI). For primary antibodies, we used rabbit anti-Epac1 antibody (1:50, ab109415, Abcam, Cambridge, UK) or polyclonal rabbit anti−human Piezo2/FAM38B antibody (1:200, LS-C180179, LSBio, Seattle, USA) or serotonin antibody (1:200, sc-58031, Santacruz Biotech, Texas, USA). For secondary antibodies, we used goat anti-rabbit IgG H&L (HRP) (1:1000, ab205718, Abcam, Cambridge, UK), and goat anti-rabbit IgG H&L (Alexa Fluor^® 488) (1:1000, ab150077, Abcam, Cambridge, UK). The samples were then viewed under a fluorescence microscope (Olympus, Japan).

The colonic tissues and DRGs were homogenized with RIPA lysis buffer. After cooling in an ice-water bath, the tissues were immediately centrifuged at 10000 rpm and 4°C for 10 minutes, and the supernatant was removed for testing. Enhanced BCA Protein Assay Kit (P0010) was used to measure protein concentrations. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk for 1 h at room temperature before being probed with rabbit anti-Epac1 antibody (1:1000, ab109415, Abcam, Cambridge, UK) or polyclonal rabbit anti−human Piezo2/FAM38B antibody (1:1000, LS-C180179, LSBio, Seattle, US) or rabbit anti-HTR3A antibody (1:500, 10443-1-AP, Proteintech, Chicago, USA) at 4°C overnight. After washing the membranes, they were subsequently incubated with goat anti-rabbit IgG (H+L) HRP (1:5000, S0001, Affinity Biosciences, Jiangsu, China) at room temperature for 1 h. Protein expression was quantified using Image J (National Institutes of Health, Bethesda, Maryland, USA).