Anti nf b p65

For immunoblotting, protein extracts (15 µg) from the thoracic aorta or total cellular proteins (50 µg) were electrophoresed on 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% dry milk in PBS-Tween buffer (0.1% Tween 20; pH 7.5) for 60 min and incubated with mouse monoclonal anti-GAPDH (TA-08, Zhongqian Jinqiao, 1/2000), rabbit anti-LOX-1 (alias: OLR1, 11837-1-ap, Proteintech, 1/1000), rabbit anti-NOX4 (Millipore, Billerica, MA, USA, 1/2000), rabbit polyclonal anti-NFĸB p65 (Santa Cruz Biotechnology, CA, USA, 1/1000); horseradish peroxidase-labeled goat anti-mouse IgG (H + L) (ZB-2305, Zhongshan Jinqiao, 1/2000), horseradish peroxidase-labeled goat anti-rabbit IgG (H + L) (ZB-2301, Zhongqian Jinqiao, 1/2000). The immunoreactive bands were detected by an enhanced chemiluminescence system (Millipore, Billerica, USA). Related signals were quantified using Scion Image Software (Scion Corp., Frederick, USA).

The fixed tissue samples were rinsed down in tap water for 10 min, dehydrated in ascending grades of ethyl alcohol, and cleared in xylene. After that, they were immersed in molten paraffin at 58–62 °C. About 5 µm histological sections were made and stained with hematoxylin and eosin. The slides were ready to examine under a bright-field microscope.
The immunohistochemical staining technique was performed by Khalil et al. [56 (link)]. Sections were dewaxed and set in a 0.05 M citrate buffer, pH 6.8 solution for antigen retrieval. Next, these sections were placed in a 0.3% H₂O₂ solution for the deactivation of endogenous peroxides followed by protein blocking. Then, sections were incubated with an anti-LC3B antibody, an autophagosome marker (cat No. ab48394, Abcam, CA, USA), and anti-NF-ĸB/P65 (cat No. sc-8008, Santa Cruz, CA, USA) at a dilution rate of 1:100. The slides were then incubated with secondary goat anti-rabbit antibody (cat No. K4003, EnVision+TM System HRP polymer; Dako, Japan) for polyclonal antibodies for 30 min at room temperature following phosphate buffered saline rinse. Slides were then covered with a drop of DAB kit solution and eventually counterstained with Mayer’s hematoxylin. The staining labeling indices of the antibodies were presented as the mean of the percent of positive area/mm².