Anti nf kb p65

The sections used for immunofluorescence staining were incubated with primary monoclonal mouse anti-MBP antibody (1:500, Abcam, Cambridge, MA), anti-GAP43 and anti-CD68/ED1 (1:100; Santa Cruz, Dallas, TX) antibodies and polyclonal rabbit anti-activated caspase-3 (1:500; Cayman Chemical, Ann Arbor, MI), anti-NF200 (1:500, Abcam, Cambridge, MA), anti-GFAP (1:200, Thermo Fisher Scientific Waltham, MA), anti-Olig1 (1:500; Abcam, Cambridge, MA), anti-CNTF (1:500, Abcam, Cambridge, MA), anti-NF-kB p65 (1:500, Abcam, Cambridge, MA) antibodies and anti-BDNF(1:500, Abcam, Cambridge, MA) antibodies overnight at 4 °C. The sections were then washed with phosphate-buffered saline (PBS) and incubated with 1:200 TRITC (rhodamine)-conjugated secondary antibodies for 1 h at 37 °C (Invitrogen, Carlsbad, CA). The sections were finally coverslipped with Antifade Gel/Mount Aqueous Mounting Media (Southern Biotech, Birmingham, AL).
Five sections from the brain cortex and anterior horns of the spinal cord for each animal were randomly selected and images were photographed under 200x magnification in three vision fields per section. GFAP, MBP, CD68-immunoreactive areas were analyzed with NIH image software, and the numbers of GAP43, caspase-3, NF-200, Olig1, CNTF, NF-kB p65, BDNF-labeled cells were counted.

About 100 mg of bladder tissue were lysed by RIPA lysate with a final concentration of 1 mM phenylmethylsulfonyl fluoride (PMSF). Protein quantification was performed using the Bio-Rad DC Protein Assay Kit (Guangzhou Yuwei Biotechnology Instrument Co., Ltd., Guangzhou, China). Each sample was added SDS loading buffer. And boiling in water for 10 min, the sample was load on a 10% SDS–polyacrylamide gel and run at 120 V, 30 min, and 100 V for 90 min. The protein was transferred from the gel to a PVDF membrane. The membrane was immersed in 1 × TBST containing 5% skimmed milk powder and gently shaken at room temperature for 2 h to block non-specific binding sites. PVDF membrane was wash 1 × TBST 3 times, 5 min/time. Add primary antibodies (anti-CB1, rabbit, 1: 250; Anti-NF-kB p65, rabbit, 1: 1000; anti-p-JNK, rabbit, 1: 1000; Abcam), incubated at 4 °C overnight, washed 3 times with 1 × TBST, 5 min/time, and then added with secondary antibody IgG (Affinity Biosciences Bio, S0001, goat anti-rabbit, 1: 20000) respectively and incubated 3 times with 1 × TBST at room temperature for 1 h. Development was performed with ECL. The gray value of Western blotting experimental protein expression was determined by Image J software, and the experiment was repeated three times.

As described by Taylor et al. [12 (link)], the total protein was first collected from cells with a RIPA lysis buffer (Solarbio) and then quantified with a BCA protein assay kit (Solarbio). After electrophoresed on SDS-PAGE, the samples were then transferred onto PVDF membranes (Millipore, MA, USA) and blocked with 5% nonfat milk for 1 h at room temperature. The primary antibody of Anti-Bax (Abcam, Cambridge, UK), Anti-Cleaved Caspase-3 (Abcam, Cambridge, UK), Anti-Bcl-2 (Abcam, Cambridge, UK), Anti-NF-kB p65 (Abcam, Cambridge, UK) and the secondary antibodies of HRP conjugated Goat-anti-Rabbit IgG (1:10,000, Invitrogen) were used for the incubation. After reacting with ECL reagent (Millipore, MA, USA), the samples were then examined in a Bio-Rad imaging system (Hercules, CA, USA). Image J software was used to measure the gray value of each sample.

Total proteins were extracted from the mesenteric lymph nodes with RIPA buffer supplemented with a cocktail of protease inhibitors, including PMSF. An equal amount of 20 μg of protein was fully electrophoresed on 10% SDS polyacrylamide gels and transferred to polyvinylidene fluoride membranes. After blocking with 5% skim milk at room temperature for 1 h, the membranes were incubated with primary antibodies overnight at 4 °C. The primary antibodies against GAPDH were obtained from Abcam and were diluted at a 1:10000 ratio, and the other antibodies were as follows: anti-TLR4 (1:1000, NOVUS), anti-S100A8/A9 (1:1000, NOVUS), anti-NF-kB p65 (0.5 μg/ml, Abcam) and anti-TNF-α (1:1000, Abcam). Subsequently, the membranes were incubated with goat anti-rabbit or anti-mouse secondary antibodies (diluted at a 1:5000 ratio), and then an ECL Detection System (Millipore) was used to detect the protein.