Acc 049

Fixed colonic mucosal samples were dehydrated, paraffin embedded, and sectioned. Tissue sections of 4 μm thickness were mounted on silicone-coated slides, deparaffinized, and heated in citrate buffer (pH 6) using high-pressure cooking for antigen retrieval. Then, the sections were incubated with primary antibodies of TRPV1 (ab3487, 1 : 50, Abcam, Cambridge, MA, USA), TRPA1 (ACC-037, 1 : 50, Alomone Labs, Jerusalem, Israel), TRPV4 (ACC-034,1 : 50), TRPM2 (ACC-043, 1 : 50), and TRPM8(ACC-049,1 : 200) at 4°C overnight. After being rinsed with phosphate buffer saline (PBS) for three times, they were incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) for 30 minutes at room temperature and visualized with a diaminobenzidine-enhanced liquid substrate system (Sigma-Aldrich, St. Louis, MO, USA). For semiquantitation, captured images were analyzed with ImageJ software (National Institutes of Health).

Cells were grown on coverslips, fixed with 4% PFA and permeabilized in 5% FBS/0.1% Triton X-100. Afterward, cells were blocked and incubated with primary antibodies overnight at 4°C (see Supplementary Table S4). After washing, cells were incubated with Alexa Fluor conjugated secondary antibodies and counterstained with DAPI. For the semiquantitative analysis of TRPM8 expression, we adopted a nonpermeabilizing protocol to detect only its localization at the plasma membrane. Cells grown on coverslips were washed with ice-cold 2% BSA in PBS and incubated with anti-TRPM8 (Alomone Labs, ACC-049) and anti-E-Cadherin antibodies for 1 h at 4 °C. After washing, cells were incubated with Alexa Fluor conjugated secondary antibodies, fixed with 4% PFA and counterstained with DAPI. For the quantification of plasma membrane associated TRPM8 channels, TRPM8 dots from more than 6000 cells were counted by two independent researchers. All the images were acquired using a Leica DM6000 CS confocal microscope. Immunofluorescence studies were performed in at least three independent biological replicates; representative data are shown.