Horseradish peroxidase hrp

Three animals were sacrificed 24 h after ischemia by rapid decapitation under deep anesthesia. Brain tissues were prepared for western blotting. Protein concentration was determined by the Bradford assay (Tiangen Biotech Co., Ltd., Beijing, China). Protein samples (50 μg per lane) were electrophoresed in 10% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) at 60 V for 2 hours at 4°C in a transfer buffer containing 48 mmol L^-1 Tris-base, 39 mmol L^-1 glycine, and 20% methanol. The blots were blocked in fresh blocking buffer (Tris-buffered saline with 0.05% Tween 20 [TBS-T] plus 5% non-fat dry milk) for 1 h at room temperature. The blots were then incubated at 4°C overnight with anti-Atf2 antibody (sc-164978), anti-c-Fos antibody (sc-52), or anti-C/EBPγ antibody (sc-25769) (all at 1:1000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and anti-β-actin antibody (1: 10000, Santa Cruz Biotechnology). A secondary antibody conjugated with horseradish peroxidase (HRP, 1:5000, Bio-Rad) was used. Immunoblots were visualized on X-ray film by a chemiluminescence reaction (Pierce, Rockford, IL). Image analysis of the blots was performed on optical density-calibrated images using AlphaEase Stand Alone software (Alpha Innotech Corp., San Leandro, CA).

Cells were lysed in gel loading buffer (Invitrogen, Carlsbad, CA, USA); samples were boiled for 10 min, and proteins were separated by SDS-PAGE. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (MilliporeSigma, Burlington, MA, USA) and probed with the antibodies anti-Vif (Aids Reagent Program; catalog number [cat. no.] 6459) and β-actin (MilliporeSigma; cat. no. A1978). An anti-mouse secondary antibody conjugated with horseradish peroxidase (HRP; Bio-Rad, Hercules, CA, USA) was visualized with the Immobilon Western HRP substrate (MilliporeSigma, Burlington, MA, USA) and detected with the Li-Cor C-DiGit Blot Scanner (Lincoln, NE, USA).

293T cells were seeded at 8 X 10⁵ cells in each well of a 6-well plate and cultured in 1.5 mL of complete DMEM medium in a 37°C incubator at 5% CO₂ overnight. The next day, lentivirus vector was added to the cells at a MOI of 2.5, 5, 10 or 20 for 48 h. For PC3 cells, in addition to lentivirus, polybrene (Sigma-Aldrich) was added to the cell medium at a concentration of 2 μg/mL. Cells were lysed in 1% NP-40 lysis buffer containing a Pierce Protease Inhibitor Tablet (Thermo Fisher Scientific). Protein lysates were prepared in 1x NuPAGE LDS sample buffer (Thermo Fisher Scientific), samples were heated for 10 min at 70°C, and proteins were separated with 4-12% NuPAGE Bis-Tris gels (Thermo Fisher Scientific). Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (MilliporeSigma) and probed with the antibodies anti-FDPS (Bethyl Laboratories Fortis Life Sciences, Waltham, MA, USA) and β-actin (MilliporeSigma). Anti-mouse or rabbit secondary antibody conjugated with horseradish peroxidase (HRP) (Bio-Rad, Hercules, CA, USA) was visualized with the Immobilon Western HRP substrate (MilliporeSigma) and detected with the LI-COR C-DiGit Blot Scanner (Lincoln, NE, USA).

Proteins (30 μg) were loaded on a precast gel in equal amounts to perform electrophoresis. After the electrophoresis, proteins on the gel were transferred to a nitrocellulose membrane which was blocked by incubating for 1 hour with 5% milk at room temperature. Primary antibodies for fibronectin(Cat: 26836), N‐cadherin(Cat: 13116), E‐cadherin(Cat: 14472), p‐p65 (Ser536)(Cat: 3033), α‐Tubulin(Cat:2125), Lamin A/C(Cat:4777), and β‐Actin(Cat: 4970) p‐IKKβ (Ser177)(Cat:2078), IKKβ(Cat:8943), p‐IκBα (Ser32)(Cat:5210), and IκBα(Cat:9242) (Cell Signalling Technology, Danvers, MA), p65(Cat:PA5‐16545), AKIP1(Cat:PA5‐106533), p‐Akt (Ser473)(Cat: OMA1‐03061), Akt(Cat:44‐609G), (Thermofisher, Massachusetts) were used during subsequent membrane incubation for 12 hours at 4°C. Following rinsing extensively with Tris‐buffered saline‐Tween 20, membranes were blotted with secondary antibodies conjugated to horseradish peroxidase (HRP) (Bio‐Rad) for 1 hour, and chemiluminescence kit was used to detect the signal intensity, which was quantified with densitometry using NIH ImageJ software (ImageJ, RRID: SCR_003070) and a Xerox scanner.

Serial 10 μm cross sections of the right muscle were obtained on a cryostat (CM510; Leica, Wetzlar, Germany) at −20°C. The sections were warmed to room temperature (RT) and then preincubated in 1% normal goat serum (EMD Millipore, Billerica, MA) in 0.1 M phosphate buffered saline (PBS; pH 7.6) at RT for 10 min. The primary monoclonal antibody was then applied: either (1) fast myosin (1 : 2000; Sigma, St. Louis, MO), which specifically reacts with the myosin heavy chain- (MHC-) IIa and IIx, or (2) SC-71 (1 : 1000; Developmental Studies Hybridoma Bank, Iowa City, IA), which specifically reacts with MHC-IIa. The sections were incubated in these primary antibodies overnight at RT and incubated with a secondary antibody (goat anti-mouse IgG) conjugated with horseradish peroxidase (HRP, Bio-Rad, Hercules, CA, 1 : 1,000) at RT for 3 hours. Diaminobenzidine tetrahydrochloride was used as a chromogen to localize HRP. Images of the stained muscle fibers were recorded with a photomicroscopic (E600; Nikon, Tokyo, Japan) image processing system (DS-U1; Nikon). The fibers were classified as Type I, IIa, or IIx/b fibers based on their immunohistochemical staining properties, and population and cross-sectional areas (CSAs) of each muscle fiber type were calculated in deep and superficial portions (Figure 1(a)).

Cells were lysed in lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 150 mM KCl, 1 mM dithiothreitol and 1% Nonidet P-40) (all from Sigma-Aldrich, Dorset, UK) containing protease inhibitors (Complete^TM, Mini Protease Inhibitor Cocktail, Merck, Life Science S.r.l., Milan, Italy), sonicated and then centrifuged at 4 °C for 20 min. The supernatant protein lysates were separated by SDS-PAGE (polyacrylamide gel electrophoresis) gradient gels (Bio-Rad, Hercules, CA, USA), following semidry blotting to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Merk-Millipore, Milan, Italy). Membranes were blocked in Tris buffered saline containing 0.1% Tween 20 (TBS) and 3% BSA (Sigma-Aldrich, Dorset, UK) before probing with the primary antibodies and then with the appropriate secondary antibodies coupled to horseradish peroxidase (HRP) (Bio-Rad, Segrate, MI, Italy). Enzymatic signal was visualized by chemiluminescence (ECL Detection system, Amersham GE Healthcare, Milan, Italy).