Horseradish peroxidase linked anti rabbit igg

Total protein was extracted from cell pellets by lysing in RIPA buffer (Sigma-Aldrich, Saint Louis, MO, USA) containing 1x protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). EZQ Protein Quantitation Kit (Molecular Probes, Eugene, OR, USA) was used to measure protein concentration. Forty micrograms of total protein were denatured and electrophoresed on Any kD^TM Mini-Protean TGX precast polyacrylamide gels (Bio-Rad, Hercules, CA, USA), electroblotted on nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), and probed with the respective antibodies against acetyl-histone H3 (Lys9; C5B11; 1:1000)) or β-actin (D6A8; 1:1000) purchased from Cell Signaling Technology (Danvers, MA, USA). The bound antibodies were visualized with horseradish peroxidase-linked anti-rabbit IgG (1:3000; Cell Signaling Technology, Danvers, MA, USA) and enhanced chemiluminescence (Amersham, Pittsburgh, PA, USA). β-actin in the corresponding cell lysates was used as an additional loading control.

In vivo expression levels of fatty acid oxidation-associated genes in CD3-positive T cells were measured RT-PCR and western blot. Briefly, tumors were extracted and digested by 1 mg mL⁻¹ collagenase (Sigma-Aldrich). Single cells were stained with APC conjugated anti-mouse CD3 antibody and CD3-positive cells were collected using BD FACSAria™ III sorter (BD Biosciences, San Jose, CA, USA). For RT-PCR, total RNA was isolated from CD3-expressing T cells using the TRIzol reagent (Thermo-Fisher Scientific). cDNA was synthesized from mRNA using an AccuPower RT PreMix. Quantitative real-time RT-PCR was performed using a LightCycler FastStart DNA Master SYBR Green I system. For western blot, CD3-expressing T cells were lysated with RIPA buffer containing protease inhibitor. The amounts of protein were quantified by BCA assay. Primary antibodies for detecting following proteins were uses: anti-mouse CPT1B (Proteintech), anti-mouse LCAD (Proteintech), anti-mouse MCAD (Proteintech) and anti-mouse β-actin (Cell signaling). Horseradish peroxidase-linked anti-rabbit IgG (Cell signaling) was used as secondary antibody. Amersham ECL Prime Western Blotting detection reagent was used for signal detection.

The freshly harvested pancreatic tissues were homogenized and lysed in ice-cold RIPA lysis buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris [pH7.5], 1 mM PMSF, 10 g/ml Leupeptin) for 60 minutes by vortexing every 5 minutes. The lysates were separated by centrifugation at 14,000×g for 10 minutes at 4°C. The supernatants were collected, aliquoted, and stored at −80°C until Western blot assay conducted. 20 µg of total protein per lane was separated by 10% SDS-PAGE gel and then were transferred to a polyvinylidene fluoride membrane. The membrane was blocked by 5% FBS in 1X Tris-Buffered Saline with 0.1% Tween-20 and then incubated with anti-ColI antibody (dilution 1∶10000, Abcam, Cambridge, MA, USA), MMP-9 antibody (1∶5000 Abcam), MMP-13 antibody (1∶10000, Abcam) and GAPDH antibody (1∶15000, Proteintech, Chicago, IL, USA) at room temperature for 90 minutes. The membrane was then incubated with diluted horseradish peroxidase-linked anti-rabbit IgG (1∶1000, Cell Signaling Technology, Boston, MA, USA) for 1 hour at room temperature. The membranes were washed for 5 minutes by 3 times between each step. The protein-antibody complex was detected by the chemiluminescent substrate (Cell Signaling), the emitted light was captured on an X-ray film, and the intensities of bands were semi-quantified by ImageJ software.

In order to collect brain tissues for the immunohistochemical and western blot analyses, mice were transcardially perfused with ice-cold PBS immediately after the FST. For each brain of 5 mice, tissue extracts were prepared as described previously [46 (link)], and equal amounts of protein (50 μg) were applied for the immunoblot analysis. The specific antibodies used in this study were rabbit antibodies against BDNF (dilution 1:1000, Abcam, Cambridge, UK) and rabbit antibodies against β-actin (1:1000, Sigma-Aldrich, St. Louis, MO, USA). The secondary antibody was horseradish peroxidase-linked anti-rabbit IgG (Cell Signaling, Denver, MA, USA). Immunoreactive bands were visualized by ECL-prime (GE Healthcare, Chalfont St. Giles, UK), and band intensities were measured using a LAS-3000 imaging system (Fujifilm, Tokyo, Japan). The quantitative analysis of band intensities was conducted using ImageJ software (NIH, Bethesda, Rockville, MD, USA), and data were normalized with β-actin.