Rabbit anti lc3 antibody

The cellular total protein were obtained by a protein extraction kit. Besides, the Bradford protein assay kit (BESTBIO) was used to determine the protein concentration. 30 μg or 50 μg of total cell extracts were analyzed by Western blotting. The proteins were loaded onto 15% SDS-PAGE gels and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). Membranes were incubated with 1 μg/ml Rabbit anti-ATG4a antibody (Polyclonal, ABGENT), 2 μg/ml mouse anti-SQSTM1/p62 antibody (Monoclonal, Abcam), 2 μg/ml rabbit anti-LC3 antibody (Cell Signaling Technology, Inc.), 2.5 μg/ml rabbit anti-GAPDH antibody (Monoclonal, Cell Signaling Technology, Inc.) or 2 μg/ml mouse anti-β-actin antibody (Monoclonal, Cell Signaling Technology, Inc.). After that, the membranes were incubated with 1 μg/ml HRP-conjugated goat anti-rabbit IgG secondary antibody or 1 μg/ml HRP-conjugated goat anti-mouse IgG secondary antibody (Proteintech Group, Inc.). Immunoreactive band analysis was performed by using the enhanced western bright ECL reagent (Advansta, United States). Densitometry analyses of western blots were performed with ImageJ software (version 1.46). Western blots were developed to be linear in the range used for densitometry. All results were expressed as a relative ratio to the β-actin or GAPHD. At least three or four independent western blot experiments were performed.

Deparaffinized myocardial tissue sections were subjected to 3% hydrogen peroxide for 15 min. For heat-induced epitope retrieval, the sections were placed in a 0.01 mol/L (pH 6.0) citrate buffer and heated at 120 °C for 10 min. Nonspecific binding was blocked by pre-incubation with 5% goat serum in phosphate-buffered saline for 20 min at room temperature. The sections were incubated with a rabbit anti-LC3 antibody (diluted 1:3200, Cell Signaling, Danvers, MA, USA) in blocking buffer for overnight at 4 °C. Phosphate-buffered saline was used to wash sections. Sections and were then incubated with streptavidin-biotin complex at room temperature for 20 min. Following extensive phosphate-buffered saline washes, sections were subjected to 2% 3,3′-diaminobenzidine in 50 mmol/L Tris buffer (pH 7.6) containing 0.3% hydrogen peroxide for 5–10 min to develop the color reaction. Sections were imaged (15 sections per rat) for LC3 positivity with an Olympus BX51 microscope/software (Olympus, Tokyo, Japan) and Image-Pro Plus v6.0 software (Media Cybernetics, Rockville, MD, USA). Two pathology experts blindly scored and analyzed five random fields from each section and plotted data using mean integrated optical density (mean IOD) = IOD/area.

Extracted proteins were quantified using a BCA Protein Assay Kit (Beyotime, Nantong, China). The protein extracts (50 μg) were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. After blocking with silk milk for 2 h at room temperature, the proteins were probed with primary antibodies and specific conjugated peroxidase-labeled secondary antibodies. The immunoreactive bands were visualized using a luminescent image analyzer (Amersham Imager 600; GE, MA, USA). Protein expression levels were determined by densitometry. All experiments were performed in triplicate. The primary antibodies used were as follows: rabbit anti-MCP-1 antibody (Cell Signaling Technology, MA, USA), rat ICAM-1 antibody (1 : 500; R&D Systems, MN, USA), rabbit anti-iNOS antibody (1 : 1000; Cell Signaling Technology), rabbit anti-LC3 antibody (1 : 1000; Cell Signaling Technology), rabbit anti-P62 antibody (1 : 1000; Cell Signaling Technology), and rabbit anti-GAPDH antibody (1 : 1000; Cell Signaling Technology).

Cell lysates with equal amounts were extracted from the heart and brain samples for Western blot analysis. Protein concentration was measured using the bicinchoninic acid method (Thermo Scientific). Samples were loaded at 12% Tris glycine gels, subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and transferred onto polyvinylidene difluoride membranes (Millipore, USA). The membrane was blocked with 5% non-fat dry milk for 2 hours and incubated with primary antibody overnight at 4°C. Immunoblotting was conducted using rabbit anti-LC3 antibody (1:1000, Cell Signaling Technology, 2775S), rabbit anti-Beclin antibody (1:1000, Cell Signaling Technology, 3495S), rabbit anti-p62/SQSTM1 antibody (1:1000, Cell Signaling Technology, 5114S), rat anti-a7nAChR (1:200 Santa Cruz Biotechnology, sc-58607), mouse anti-GAPDH antibody (1:1000, Nakasugi Jinqiao). The membranes were then incubated with species-appropriate secondary antibodies for 1 hour at room temperature. The protein bands were visualized with a Bio-Rad Gel Imager.