Anti trpv1

Paraffin-embedded colon sections from patients were subjected to immunohistochemical staining using anti-TRPV1 (Santa Cruz) at a dilution of 1:50. Colon sections from mice were stained with hematoxylin and eosin (H&E). Colitis activity score was determined based on previously published criteria²⁸ (link) by a pathologist blinded to the experimental groups. In brief, the total score ranged from 0 to 14, which represents the sum of scores from 0 to 4 for inflammation severity (0, none; 1, mild; 2, moderate; 3, severe), inflammation extent (0, none; 1, mucosa; 2, submucosa; 3, transmural), crypt damage (0, none; 1, basal 1/3; 2, basal 2/3; 3, crypt lost, surface epithelium present; 4, crypt and surface epithelium lost), and percent involvement (0, 0%; 1, 1%–25%; 2, 26%–50%; 3, 51%–75%; 4, 76%–100%). NFATc2 immunohistochemical analysis was performed by using anti-NFATc2 (Santa Cruz) at a dilution of 1:100. Integrated optical density (IOD) values were measured by Image-Pro Plus 6.0 image processing software (Media Cybernetics).

Animals were sacrificed by decapitation and SNc area was rapidly removed and frozen on dry ice, and stored at −80 °C. Protein lysate was obtained by re-suspending SNc in ice-cold lysis buffer (CelLytic, Sigma, USA) containing diluted phosphatase (1:10, Roche, Monza, Italy) and protease inhibitors (1:25, Roche, Italy). After centrifugation, the supernatant was collected and protein concentration was measured using a Bicinchoninic Acid Protein Assay (Sigma, USA). Protein lysates were run on 10% gels, transferred onto nitrocellulose membranes (Biorad, USA) and western blot was performed. Membranes were blocked (Odyssey blocking buffer, LiCor, USA) and incubated overnight with the following primary antibodies: anti-GFAP (1:2000, Sigma, USA, G3893), anti-TRPV1 (1:500, Santa Cruz, USA); anti-CNTF (1:500, Millipore, Germany, MAB338). As secondary antibodies, anti-mouse 1:10000 secondary IgG HRP-Conjugated were used. Image analysis of western blots was performed using Azure 600 azure biosystems and software (LiCor, Biosciences, Rockville, MD, USA) and signal was normalized with the corresponding GFAP signal.