Total RNA was extracted from anterior eye segment samples (cornea plus conjunctiva) and trigeminal ganglia (TG) of sham and DE rats using an RNA kit (Absolutely RNA; Agilent Technologies, La Jolla, CA, USA). cDNA was synthesized from 300 ng of each sample using a cDNA synthesis kit (iScript; Bio-Rad Laboratories, Hercules, CA, USA). qPCR was performed in triplicate on 2 μL cDNA with a DNA engine (Chromo4; Bio-Rad Laboratories) using iQ SYBR Green Supermix (Bio-Rad Laboratories). PCR conditions were as follows: an initial denaturation at 95°C for 3 minutes, followed by 40 cycles of 95°C for 10 seconds, 58.5°C for 20 seconds, and 72°C for 30 seconds. Data were analyzed using the delta CT method against two reference genes (GAPDH and UBC). Primer sets were as follows: GAPDH: F: 5′-agacagccgcatcttcttgt-3′, R: 5′-cttgccgtgggtagagtcat-3′. TRPV1: F: 5′-ctgctcctggacgttgcccg-3′, R: 5′-ccagcgtcatgttccgccgt-3′, TRPM8: F: 5′aggacttggcagaacagcta-3′, R: 5′aggaaattctggaccccagc-3′. A melting curve was employed to ensure amplicon fidelity.
Samples were homogenized in 0.5 mL cold lysis buffer (1% Triton X-100, 10 mM EGTA, 10 mM EDTA, TBS pH 7.4, protease inhibitor cocktail [complete mini; Roche Diagnostic Operations, Indianapolis, IN, USA]). Homogenates were centrifuged at 4°C for 10 minutes at 12,000 g, and the supernatant retained. Protein concentration was determined by BCA assay (Pierce Biotechnology, Inc., Rockford, IL, USA), and 25 μg protein was separated on 7.5% polyacrylamide gels and transferred to a 0.45 μM nitrocellulose membrane (Bio-Rad Laboratories). Membranes were blocked and incubated at 4°C overnight in TRPV1 receptor antibody (1:1000, lot# ACC029AN0550; Millipore, Billerica, MA, USA) or TRPM8 antibody (1:1000, lot# SA2318285; Invitrogen, Waltham, MA, USA) followed by goat anti-rabbit IRDye 680 (LI-COR, Lincoln, NE, USA). Proteins were visualized using an infrared scanner (Odyssey; LI-COR) and arbitrary optical density was determined. Normalizing controls were utilized by simultaneous staining with beta-tubulin antibody (Santa Cruz Biotechnology, Dallas, TX, USA) followed by goat anti-mouse IRDye 800 secondary antibody (LI-COR).
Immunoprecipitation was performed to confirm and compare results from western blots using a commercial kit (Pierce Classic IP Kit; Thermo Fisher Scientific, Rockford, IL, USA). Briefly, anterior eye and TG samples (150 mg) from sham and DE rats were homogenized and incubated with 2 μg of antibody for TRPV1 and TRPM8 as noted above. Protein-antibody complexes were separated from homogenate with protein A/G agarose, dissociated with loading buffer and separated on an 8% to 20% precast gel (Protean TGX; Bio-Rad Laboratories) in a mini cell (Mini Protean Tetra Cell; Bio-Rad Laboratories). The gel was removed and stained with a commercial stain (GelCode Blue Safe Protein Stain; Thermo Fisher Scientific) before imaging.