Bx ura2 camera

At different time points after surgery, mice were anesthetized intraperitoneally using sodium pentobarbital (100 mg/kg) and killed by swift decapitation according to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health. The mouse hearts were fixed in 4% paraformaldehyde overnight at 4°C and then dehydrated through a graded ethanol series, cleared in xylenes, and embedded in paraffin wax. Samples were subsequently cut into 4 μM thick sections and stained with HE. For immunohistochemical studies, the sections were treated with 3% hydrogen peroxide for 15 min to block endogenous peroxidase activity. Then, sections were sealed with normal goat serum and incubated at 4°C overnight with anti-IKKε antibody (1 : 200 dilution, Cell Signaling Technology #2690). The secondary antibody was horseradish peroxidase- (HRP-) conjugated goat anti-rabbit IgG (Beijing Zhongshan Biotechnology Co., Beijing, China). The signal was revealed by using a DAB Substrate Kit (Beijing Zhongshan Biotechnology Co.). Nuclei were counterstained with hematoxylin. Images were captured using an Olympus BX-URA2 camera with NIS-Elements D 3.2 software.

Tissues collected for morphological analysis with aortic roots and ascending aorta were prepared for OCT-embedded cryosections. The sections were fixed in 4% parafor maldehyde for 30 min, washed in PBS, and then incubated in buffered normal goat serum to prevent non-specific binding of antibodies for 1 h at room temperature. The sections were then incubated separately overnight with antibodies against CD68 or LPL (1:100; Santa Cruz Biotechnology, CA, USA), followed by incubation with Alexa Fluor 592-conjugatged goat anti-rabbit IgG (1:200;Invitrogen, Carlsbad, CA, USA) for 1 h at 37°C in a humidified box. Thereafter, the sections were washed in PBS and counter-stained with Hoechst dyetostain DNA and illuminate the nuclei. Photomicrographs were taken at random using an Olympus BX-URA2 camera in 4 sections per mouse sample.

Immunohistochemistry staining was performed on formalin-fixed kidney tissues. Sections were deparaffinized with xylene and rehydrated in a graded alcohol series and then placed in PBS (pH 7.5). The microwave antigen retrieval procedure (citrate buffer, pH 6.0) was performed for 10 min. After that, sections were immersed in 3% hydrogen peroxide for 10 min to block endogenous peroxidase, then treated with 3% BSA (diluted in PBS) for blocking nonspecific binding sites, and incubated overnight at 4°C with the following primary antibodies: rabbit anti-mouse ki67 (1:2000, Abcam), IKKα (1:200, Abcam), NIK (1:200, Santa Cruz Biotechnology), p52 (1:200, Santa Cruz Biotechnology), RelB (1:300, Cell Signaling), IL10 (1:500, Abbiotec) and Foxp3⁺ (1:100, Santa Cruz Biotechnology). The next day, these slides were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-rat secondary antibody for 1 h at room temperature. 3,3-Diaminobenzidine tetrahydrochloride was applied to the slides for developing brown color. Counterstaining was carried out with Eosin. All slides contained duplicate sections, from which one served as a control for secondary antibody binding specificity. The positive areas were measured in five randomly chosen fields, and quantified blindly using an Olympus BX-URA2 camera.