Ab23722

We followed the methods of Wang et al. [21 (link)]. After blocking, the aorta sections were incubated overnight at 4°C with a rabbit anti-AGE antibody (1 : 200, ab23722, Abcam, Cambridge, UK), rabbit anti-RAGE antibody (1 : 200, ab3611, Abcam, Cambridge, UK), rabbit anti-Nox4 antibody (1 : 150, ab133303, Abcam, Cambridge, UK), mouse anti-3-nitrotyrosine antibody (1 : 200, ab61392, Abcam, Cambridge, UK), rabbit anti-NF-κB p65 antibody (1 : 200, D14E12, CST, Beverly, MA, USA), or mouse anti-Glo1 antibody (1 : 200, MA1-13029, Invitrogen, Waltham, MA, USA). The sections were washed and then incubated with a secondary antibody (1 : 200, peroxidase-conjugated anti-rabbit (ZB-2301) or anti-mouse (ZB-2305) antibody, ZSGB-BIO, Beijing, China) for 1 hour. Color was developed using DAB. Images were captured using an Olympus DP71 microscope. The mean IOD of staining (IOD/area) from 5 random fields on one section was assessed using Image-Pro Plus 6.0 software.

At sacrifice, blood was collected for analysis of fasting glucose (Aimstrip Plus Glucose Meter; Germaine Laboratories, San Antonio, TX, USA) and serum AGE quantification. Total serum AGEs were measured with a competitive ELISA (developed in our laboratory) using an anti-AGE antibody (Abcam, Cambridge, MA, USA; ab23722). For this study, 1 unit (U) corresponds to 3 mg AGE-BSA.

RAW264.7 cells (8.0 × 10⁵ cells) were incubated for 1 h on 35-mm dishes, washed in buffer, and treated with 200 μg/ml BSA, AGE2, or AGE3. Thereafter, cells were rinsed with 0.1 M PBS and fixed with 4% PFA phosphate buffer solution for 10 min. After washing in 20 mM Tris-HCl pH 7.6, the cells were incubated in 1% BSA/10% normal goat serum/0.3 M glycine in PBS-T for 1 h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with rabbit anti-AGE (BSA-AGE and HSA-AGE) polyclonal Ab (Ab23722, Abcam) overnight at 4 °C. The secondary Ab of labelled polymer combined with peroxidase and anti-rabbit IgG (H + L) (GHP516G, Biocare Medical, Pacheco, CA, USA) was applied for 1 h, and AGE protein localisation visualised with 3, 3′-diaminobenzidine (DAB) chromogen system (Dako, Carpenteria, CA, USA). After rinsing with 0.1 M PBS, cells were post-fixed with 2% (w/v) osmium tetroxide for 1 h, washed with 0.1 M PBS, dehydrated in an ascending ethanol series (50, 70, 80, 90, 95, and 99.5%) for 5 min each and dehydrated in 100% ethanol for 30 min. The samples were embedded in an epoxy resin at 60 °C for 1 day. Ultrathin sections were cut, stained with lead citrate, and observed using a TEM HT-7700.