Ab172730 rabbit monoclonal igg

After the electrophysiology studies, the brain was rapidly removed, and samples were immersed in 10% formalin for 24 h, embedded in paraffin and cut into 5-μm thick slices. The paraffin sections were deparaffinized, rehydrated and soaked in 0.1 M of citric acid buffer for 15 min at 92–98°C in a microwave oven, and washed with PBS. Then, the sections were incubated with the primary antibodies of anti-HMGB1 (ab172730-Rabbit monoclonal IgG, 1:5,000; Abcam) overnight at 4°C. Subsequently, the samples were incubated with horseradish peroxidase-conjugated secondary antibodies of rabbit anti-sheep IgG (KPL, Gaithersburg, MD, USA) and goat anti-mouse IgG (Zhongshan Golden Bridge Biotechnology) for 1 h at 37°C. Immunore-activity was developed with 3,3′-diaminobenzidine tetrahydrochloride (Zhongshan Golden Bridge Biotechnology). Finally, the sections were counter-stained with hematoxylin, mounted and examined by microscopy.

The expression of HMGB1 and p-ERK in the PVN were detected by Western blot analysis. For immunoblot analyses, proteins were isolated using a protein extraction kit (Beyotime Institute of Biotechnology, Jiangsu, China). The extracted protein was measured using a BCA protein assay reagent kit (Pierce, Madison, WI, USA). Equal amounts of total protein (80 μg of protein/lane) were resolved on 5–10% SDS-PAGE gels, and transferred onto polyvinylidene difluoride membranes. Then, the membranes were blocked with 5% non-fat dry milk in phosphate buffered saline (PBS) containing 0.05% Tween 20, and incubated overnight at 4°C with the respective primary antibodies against HMGB1 (ab172730-Rabbit monoclonal IgG; 1:20,000; Abcam) and primary antibodies against p-ERK (1:2,000; 9101; Cell Signaling Technology). Next, the membranes were incubated with horse-radish peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibodies (1:10,000; Zhongshan Golden Bridge Biotechnology). The blots were developed using an enhanced chemiluminescence detection kit (Millipore, Billerica, MA), and visualized using a FluorChem E Imager (Protein-Simple, Santa Clara, CA). The densities relative to β-actin were analyzed using NIH ImageJ software.