Anti age antibody

After treatment with H₂O₂ (3% in ethanol, room temperature, 15 min.) and proteinase K (100 µg/ml, 100 µl, 37 °C, 20 min.), the sections were incubated with 5% BSA-PBS buffer (room temperature, 30 min.) in order to block non-specific staining. Afterwards, the sections were incubated with the primary anti-AGE antibody (abcam, 1:100, diluted in 1% BSA-PBS), or normal mouse IgG (250 µg/ml) pre-treated with excessive CML (1:250, diluted in 1% BSA-PBS, negative control) over night at 4 °C. The sections were then washed and incubated with LINK (biotinylated anti-rabbit and anti-mouse immunoglobulin) and STREPTAVIDIN PEROXIDASE (streptavidin conjugated with horseradish peroxidase) respectively at room temperature for 10 min (both are part of reagents of LSAB2 System-HRP, products of Dako Company, Denmark). Then the peroxidase activity was visualized by incubating the sections in substrate working solution containing hydrogen peroxide and 3, 3′-diaminobenzidine tetrahydrochloride at room temperature for 5 min. The sections were rinsed for 10 min, counterstained with Mayer Haematoxylin for 1 min, treated in HCl-ethanol for 3 sec, and dehydrated in 80%, 90%, 95%, 100% ethanol for 3 sec, respectively. Then the slides were immersed in xylene for 15 min two times and mounted.

AGEs were removed from diabetic serum using an immunoadsorption method. To immunoprecipitate AGE-modified proteins, 500 μl of diabetic serum was incubated for 1 h with 25 μl of Pierce NHS-activated magnetic beads (Thermofisher Scientific) covalently conjugated with 10 μg of anti-AGE antibody (Abcam, see Supplementary Table S1 for antibodies in Additional file 1), according to the manufacturer instruction. To confirm the efficiency of AGE depletion, AGE concentration in both treated (unbound serum fraction) and untreated diabetic serum was evaluated in triplicate by ELISA (OxiSelect™ Advanced Glycation End-Product Competitive ELISA Kit, no. STA-817, Cell Biolabs, Inc., San Diego, CA, USA). Following this procedure, the concentration of AGEs in diabetic serum was reduced by about 60%, reaching a concentration similar to that of the non-diabetic serum (see the “Results” section).