Ab92544

We performed immunofluorescence staining on brain tissue sections and HBMECs. The tissue sections were dewaxed with xylene and then rehydrated in ethanol. High glucose‐treated HBMECs were fixed on cell slides with 4% paraformaldehyde. The fixed sections and cell slides were transferred to blocking solution (goat serum) for 1 h. The blocked HBMECs were incubated overnight at 4°C with primary antibodies against SREBP1 (AF6283, diluted 1:100, Affinity Biosciences) and p‐mTOR (cst5536, diluted 1:100, Cell Signaling Technology). The tissue sections were incubated overnight at 4°C with primary antibodies against SREBP1 (AF6283, diluted 1:200, Affinity Biosciences), LRP1 (ab92544, diluted 1:500, Abcam), Raptor (DF7527, diluted 1:200, Affinity Biosciences) and CD31 (ab182981, diluted 1:2000, Abcam). Immunoreactions were observed using CY3‐conjugated affinity‐pure goat anti‐rabbit IgG (BA1032, diluted 1:100, Bost Biotech) and FITC‐conjugated affinity‐pure goat anti‐mouse IgG (BA1011, diluted 1:100, Bost Biotech). Nuclei were stained with Hoechst (Invitrogen). Antifluorescent quenchers were added, and the slides were observed under a microscope (Olympus CX31 microscope).

The expression level of LRP1 and APP were measured by Western blot. Briefly, protein extracts were obtained from whole cell lysates and quantified with the Pierce BCA assay from ThermoFisher Scientific (Waltham, MA). For SDS-PAGE 10% gels were used, 10 μg of protein sample was loaded and electrophoresis was run at 150 mV for 70 min. Protein transfer to a previously methanol-activated PVDF membrane was carried out at 400 mA for 1 h at 4 °C. Membranes were blocked in blocking buffer (5% milk in TBS-Tween20) for 1 h at room temperature with shaking. Overnight incubation with primary antibodies in blocking buffer was done at 4 °C with shaking. After four 5-min TBS-Tween20 washes, membranes were incubated with secondary antibodies in blocking buffer for 4 h at room temperature with shaking. After another round of four 5-min TBS-Tween20 washes, membranes were developed with X-ray films and ECL reagent from Santa Cruz Biotechnology (Heidelberg, Germany). The antibodies and dilutions used were as follows: LRP1 1:20,000 (Abcam #ab92544) (Cambridge, UK), APP 1:1,000 (Abcam #ab15272), GAPDH 1:2,000 (Santa Cruz #sc-47724), Beta-actin 1:5,000 (Abcam #ab8227), HRP-secondary goat anti-rabbit 1:5,000 (Abcam #ab6721), HRP-secondary goat anti-mouse 1:5,000 (Dako-Agilent Technologies #P0447) (Santa Clara, CA).

Anesthetized mice were perfused transcardially with PBS, followed by 4 % paraformaldehyde (PFA) prepared in PBS. Tissues were post-fixed for at least 24 h in 4 % PFA, and transferred to 20 % sucrose solution before sectioning. Cryosections (40 μm) were permeabilized in 0.5 % PBS-Tween for 15 min and washed twice with PBS. After blocking in 5 % serum in PBS for 2 h at room temperature, sections were incubated overnight at 4 °C with antibody against Iba1 (ab5076, 1:400, Abcam), LRP1 (ab92544, 1:250, Abcam) and CD68 (14-0688, 1:50, eBioscience). After washing, sections were incubated with secondary antibodies conjugated to Alexa488 or Alexa647 (Life Technologies). Sections were mounted with Prolong Gold Anti-Fade Reagent containing DAPI (Life Technologies). All sections were imaged with a Leica TCS SP8 confocal microscope and analyzed with ImageJ. Sholl analysis was performed as described [24 (link)].

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.