Ab3611

Sections were fixed using 4% paraformaldehyde and blocked with 10% goat serum at 37°C. The primary antibody was incubated overnight at 4°C, and the secondary antibody was incubated for 1 h at 37°C following the day. TSA was incubated at 37°C for 30 min. After washing with PBS, the fixation step, serum blocking, and antibody incubation were repeated. After final fixation, the cells were stained with DAPI. Anti-fluorescence quencher containing Hoechst33342 (P0133, Biyuntian) was used to seal tablets. The primary antibodies were rabbit anti-LRP1 (1:200, ab92544; Abcam), rabbit anti-RAGE (1:200, ab3611; Abcam), and rabbit anti-CD31 (1:2,000, ab182981; Abcam). The second antibodies were horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:300, 074-1506, KPL) and HRP-conjugated goat anti-mouse IgG (1:300, 074-1806, KPL). Photographs were taken using a Pannoramic DESK, P-MIDI (3D HISTECH, Hungary) scanner. The percentage of positive areas was analyzed using ImageJ software. ImageJ was used to analyze LRP1-CD31 co-localization area, CD31 expression area, RAGE-CD31 co-localization area, and CD31 expression area in the hippocampus and cortex, respectively, and the ratio of co-localization area to CD31 area represents the relative amount of positive protein expressed per unit endothelial cell.

Paraffin-embedded tissues were cut into 4 μm-thick sections, and incubated with a primary antibody, followed by enzyme-labelled secondary antibody. The primary antibodies used were: NMDAR1 (1:100, ab68144, Abcam), BDNF (1:500, ab216443, Abcam), AGE (1:100, ab23722, Abcam), and RAGE (1:20, ab3611, Abcam). After incubation with a secondary antibody, the sections were visualized by using 3,3′-diaminobenzidine (DAB). Images were photographed and analyzed with Digital Pathology Solutions (Scanscope CS, Aperio, USA).

Cells were analyzed for human FL-RAGE expression by WB as already described [35] (link) using a goat polyclonal antibody against the extracellular domain of human RAGE (α-RAGE N-term1, 1 µg/ml; cat. AF1145, R&D Systems, Minneapolis, MN, USA) that recognizes rat RAGE as well. For detection of RAGE in rat lung and R3/1 cells lysates two additional different rabbit polyclonal antibodies against the extracellular (α-RAGE N-term2, 0.4 µg/ml; cat. sc-5563, Santa Cruz Biotechnology, California, USA) and the intracellular (α-RAGE C-term, 1 µg/ml; cat. ab3611, Abcam, Cambridge, UK) domains of human RAGE were used. Both antibodies recognize rat RAGE as well.
The membranes were blocked in TBST (10 mM Tris, pH 7.4; 0.5 mM NaCl; 0.1% Tween 20) containing 5% powdered skimmed milk for 1 hour (h) at rt. The blots were first probed with the indicated primary antibodies diluted in TBST with 5% powdered skimmed milk over night at 4°C, and then with horseradish peroxidase-conjugated anti-rabbit (1∶5000; cat. NA9340V, GE Healthcare) or anti-goat (1∶5000; cat. sc-2020, Santa Cruz Biotechnology) secondary antibodies. Proteins were visualized by an enhanced chemiluminescence (ECL) detection system (cat. RPN2106, GE Healthcare). An antibody agaist GAPDH (0.4 µg/ml; cat. sc-25778, Santa Cruz Biotechnology) was used on the same membranes after stripping and served as loading control.

The kidney and pancreas were fixed with 10% formalin solution and embedded in paraffin. The paraffin block was cut into 5 μm thick and stained with hematoxylin and eosin (HE). After rehydration, the samples were transferred to citrate buffer (pH 7.6), heated in a microwave oven at 65°C for 20 min, then incubated with rabbit anti-RAGE antibody (Abcam, ab3611, GR316801-2) and secondary antibody. The slides were dyed with hematoxylin and analyzed with a digital camera and ImagePro Plus software.

Cell extracts were prepared with a lysis buffer according to the manufacturer's instructions. Forty-microgram proteins were separated in 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with NOX-1 (ab55831, Abcam, USA), NOX-2 (ab80508, Abcam, USA), NOX-4 (ab154244, Abcam, USA), RAGE (ab3611, Abcam, USA), or GAPDH (ab8245, Abcam, USA) antibodies at 4°C overnight and subsequently with peroxidase-conjugated secondary antibody for 1 h at room temperature. Proteins were visualised using electrochemiluminescent reagents.

The human osteosarcoma tissue microarray OS804d was purchased from US, Biomax Inc. containing 40 cases of osteosarcoma. We performed IHC staining using a commercial IHC kit (cat. no. ab64264, Abcam, Cambridge, U.K.), following the manufacturer's procedural guidelines. Tissue sections were removed the wax using Sub-X and then rehydrated with a series of ethanol solutions. Antigen retrieval was performed by incubating slides with protease, and then blocking with protein block reagent for 10 minutes. The CML (cat. no. ab125145, Abcam) and RAGE (cat. no. ab3611, Abcam) antibodies were incubated with the sections overnight at 4°C. HRP-conjugated secondary antibody was then applied, and the sections were treated with DAB substrate. Subsequently, the slides were counterstained with hematoxylin, and sealed with mounting medium. The stained sections were analyzed using the IHC profiler plugin in ImageJ software. A score of 0 indicated negative staining, score of 1 indicated low positive staining, score of 2 indicated positive staining, and score of 3 indicated high positive staining.