Anti tnf α

The hepatic tissue was homogenized into cold lysis buffer and the lysate was centrifuged and the supernatant was collected in a fresh tube supplemented with protease inhibitor cocktail (Himedia, India) and kept at −80 °C.³² The protein samples were heat denatured and separated by SDS-PAGE followed by electrotransfer to the nitrocellulose membrane. The membranes were blocked in blocking buffer and then exposed to specific primary antibodies diluted in the blocking buffer and kept overnight at 4 °C. The primary antibodies used were rabbit polyclonal anti-phospho AMPK, anti-AMPK, anti-IL-6 and anti-TNF-α (Abcam, USA). The next day membrane was washed and subjected to incubation for 1 h with anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase (Thermo Fisher Scientific, USA) diluted in the same blocking buffer. After a series of washes, they were exposed with the chemiluminescent ECL substrates (Thermo Scientific, USA) and the desired protein bands were detected by chemiluminescent gel documentation system (Bio-Rad, USA). β-Actin was used as a housekeeping protein for equal amounts of protein loaded into the gel. The protein bands were densitometry quantified by Image J, software (NIH, USA) and data presented as arbitrary units.

Immunocytochemistry (ICC) was performed as previously described.³⁴ (link) The cells were grown in chamber slides. The hCECs were fixed in ice-cold 4% paraformaldehyde for 15 minutes 1 day after infection and then permeabilized with 0.3% Triton X-100 (Sigma-Aldrich) for 5 minutes and blocked in 3% BSA for 30 minutes. The primary antibodies were anti-CMV, clone 8B1.2 (1:2000; Sigma-Aldrich); anti-ZO-1 (1:100; Abcam, Cambridge, UK); anti-TNF-α (1:500; Abcam); and anti-nuclear factor kappa B (NF-κB) (1:100; Sigma-Aldrich). Alexa Fluor 488 and 594 secondary antibodies (1:1000) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). We further determined if changes induced by CMV infection were suppressed by ROCK inhibitors (Y27632, K115), because the Rho–ROCK pathway is a common downstream component of TNF-α signaling.

Stomach tissues obtained from mice were prepared, stained, and imaged to assess cytokine expression. The primary antibodies used for immunofluorescence staining were: lectin GS II, anti-VEGF-β, anti-IL-10 (1:200; Santa Cruz Biotechnology), anti-IFN-γ (1:50; Abcam, Cambridge, UK), anti-IL-6 (1:500; Abcam), anti-TNF-α (1:100; Abcam), anti-IL-1β (1:100; Abcam), and anti-proteinase 3 (Santa Cruz Biotechnology). Expression levels of VEGF-β, GS II, and cytokines were evaluated under confocal microscopy after immunofluorescent staining of deparaffinized tissue sections.

To determine IL-1β, IL-6, and TNF-α levels, the L4–L6 segments of the spinal cord were collected 4 h after intrathecal injection on day 12 postinoculation. The tissues were washed twice with PBS and cut into pieces with sterile scissors. After being milled, the tissues were digested with 1% collagenase for 25 min at 37°C. The supernatant was collected after centrifugation (12,000× g, 10 min). The samples were then boiled at 95°C for 10 min before being processed for Western blot analysis. Primary antibodies anti-IL-1β, anti-IL-6, and anti-TNF-α (rabbit; Abcam), together with the antirabbit secondary antibody, were used to detect the cytokines produced in these tissues.

Frozen specimens from rodents were homogenized in 100 μl lysis buffer containing a mixture of proteinase and phosphatase inhibitors and then centrifuged at 15 000 rpm for 15 min at 4°C. The protein concentration was determined using a BCA Protein Assay Kit (Pierce, Rockford, IL). A total of 50 μg of protein was resolved on 12% precasted SDS‐PAGE gels, then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA). The PVDF membrane was blocked with 5% non‐fat milk in PBS containing 0.1% Tween 20 for 2 h at room temperature and then incubated overnight at 4°C with primary antibodies. The following antibodies were used in this study: GAPDH, anti TNF‐α, anti‐ASIC1, and anti‐ASIC3 antibody (1:1000, abcam, USA). After washing with Tris‐Buffered Saline, 0.1% Tween 20 Detergent (TBST), the blots were incubated for 2 h at room temperature with HRP‐conjugated secondary antibody (1:5000; Amersham Biosciences, San Francisco, CA, USA), visualized by using Electro‐Chemi‐Luminescence (ECL) chemiluminescent detection system (Amersham Biosciences).

Naloxone was purchased from Taizhou Hongyao Chemical Co., Ltd. Naltrexone and phloroglucinol were supplied by Aladdin. The dialysis bags, FITC, fura-2AM fluorescent probe, nissl staining solution, hematoxylin eosin (HE) staining kit and BCA protein concentration determination kit were ordred from Beyotime Biotechnology. LPS, Fetal bovine serum (FBS), DMEM, penicillin-streptomycin (PS) and trypsin-EDTA (0.25%) were purchased from Gibco Company. Protein lysate (RIPA), phenylmethanesulfonyl fluoride (PMSF) and 4′,6-diamidino-2-phenylindole (DAPI) were bought from Beijing Solarbio Science & Technology Co., Ltd. Anti-CD11b, anti-F4/80, anti-iNOS, anti-TNF-α, anti-IL-1β, anti-IL-6, anti-IL-10, anti-Bax, anti-caspase 3, anti-bcl-2, anti-β-tubulin, anti-GAPDH, anti-β-actin were supplied by Abcam.