Trpv1

The human PC cell lines AsPc-1 and Panc-1 were obtained from the American Type Culture Collection (Manassas, VA) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic in a humidified 5% CO₂ atmosphere at 37°C. Antibodies against SHH, SMO, PTCH, Gli1, Gli2, NGF, BDNF, GDNF, TRPV1, SP and CGRP were purchased from Abcam, USA. Recombinant sonic hedgehog, NGF and BDNF were obtained from R & D Systems (Minneapolis, MN). Capsaicin was purchased from Sigma, USA.

Anti-active-β-catenin and β-catenin antibodies were purchased from Millipore (Temecula, CA, USA). Anti-CD105-PE, anti-CD146-PE, anti-CD90-PE, anti-CD34-PE, and anti-CD45-PE antibodies were purchased from BD Bioscience (San Jose, CA, USA). Unconjugated anti-GSK3β and anti-phospho-GSK3β antibodies were purchased from Cell Signaling Inc. (San Francisco, CA, USA). Anti-β-actin antibody was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Unconjugated anti-CBS, CSE, DSPP (DSP)  and TRPV1 were purchased from Abcam Inc. (Cambridge, MA, USA).

Tissue was immunostained as described previously (Bráz et al., 2012 (link)). Antibodies used included ATF3 (rabbit, 1:2 k, Santa Cruz Biotechnology), Fos (rabbit, 1:5 k, Oncogene), Iba1 (rabbit, 1:1 k, Wako), NeuN (mouse, 1:5 k, Sigma), β-Gal (chicken, 1:10 k, Abcam), TRPV1 (guinea pig, 1:5 k, generous gift of the David Julius lab), NF200 (mouse, 1:20 k, Sigma), CGRP (mouse, 1:10 k, Sigma), tyrosine hydroxylase (rabbit, 1:5 k, Millipore), and biotinylated IB4 (goat, 1:500, Vector Labs). Fluorescent secondary antibodies were used at a 1:1 k dilution; streptavidin-conjugated fluorophore was used at 1:5 k.

Primary antibodies of NLRP1, TRPV1, caspase-1, IL-6, and TNF-α were purchased from Abcam (San Francisco, CA, USA). Primary antibodies of ASC, IL-1β, and IL-18 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Capsazepine was obtained from Sigma-Aldrich (St. Louis, MO). Other general agents were commercially available.

The lumbar spine or DRG samples were dissected from mice and then were fixed in 10% buffered formalin (4°C, 24 h). The samples of the lumber spine were decalcified by 0.5 M ethylenediamine tetraacetic acid at 4°C for 3 weeks, and the L2 DRGs were dehydrated by 30% sucrose at 4°C for 48 h. The spine samples were embedded in optimal cutting temperature compound (OCT) or paraffin. The DRG samples were embedded in OCT. We used the 4 μm thick sections (lumber spine) for safranin O and fast green staining. 40 μm thick sections of the spine samples were used for nerve fiber-related immunostaining. 10 μm thick sections of the spine or DRG sample were used for other immunostaining. For immunofluorescent staining, we incubated the sections (lumber spine or DRG) with primary antibodies to CGRP (1 : 100, Abcam, U.S.), COX2 (1 : 100, Abcam, U.S.), EP4 (1 : 100, Abcam, U.S.), and TRPV1 (1 : 200, Abcam, U.S.) (4°C, overnight). Then, we incubated the sections (lumber spine or DRG) with secondary antibodies (room temperature, 1 h, avoiding light). The fluorescence or confocal microscopes were used to capture the images of spine or DRG samples. ImageJ software (National Institutes of Health, U.S.) was used for the quantitative analysis.