Ab12267

Protein expression levels in brain capillary crude membranes were analyzed by Western blotting as previously described [9 (link), 25 (link), 36 (link)]). Western blotting was performed using the Invitrogen NuPage™ Bis–Tris electrophoresis and blotting system (Invitrogen, Carlsbad, CA, USA). After electrophoresis and protein transfer, membranes were blocked and incubated overnight with the primary antibody as indicated (P-gp: 1:100 (1 µg/ml; MA126528; RRID:AB_795165; ThermoFisher, Waltham, MA, USA); β-actin: 1:1000 (1 µg/ml; ab8226; RRID: AB_306371, Abcam, Cambridge, MA, USA); hAβ40: 1:500 (1 µg/ml; ab12265; RRID:AB_298985, Abcam, Cambridge, MA, USA); hAβ42: 1:500 (1 µg/ml; ab12267; RRID:AB_298987, Abcam, Cambridge, MA, USA). Next, membranes were washed and incubated for 1 h with horseradish peroxidase-conjugated ImmunoPure^® secondary IgG (1:10,000; 0.25 µg/ml; Pierce, Rockford, IL, USA). Proteins were detected with SuperSignal^® West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA) and visualized with a BioRad Gel Doc 2000™ gel documentation system (BioRad, Hercules, CA, USA).

Dissected brains were fixed in 4% paraformaldehyde at 4°C overnight. For immunostaining using anti-Aβ antibody 4G8 (Covance) or rabbit polyclonal anti-Aβ_1–42 antibody ab12267 (AbCam), brains were pre-treated with 70% FA for 10 min. For immunostaining using aggregated-Aβ-specific antibody 7A1a (New England Rare Reagents), FA pre-treatment was not included. Primary antibody immunostaining was detected using Alexa Fluor-555-conjugated secondary antibody (Invitrogen). For immunostaining with anti-Rab5 antibody (AbCam), the secondary antibody used for detection was Alexa Fluor-647-conjugated goat anti-rabbit IgG (Invitrogen). Samples were observed and images collected using confocal microscopy (Zeiss LSM 510).

Antibodies to human Aβ40 (ab12265; RRID:AB_298985), human Aβ42 (ab12267; RRID:AB_298987), and β-actin (ab8226; RRID: AB_306371) were purchased from Abcam (Cambridge, MA, USA). C219 antibody against P-gp was from ThermoFisher (MA126528; RRID:AB_795165; Waltham, MA, USA). Complete^™ protease inhibitor was obtained from Roche (Mannheim, Germany). Fluorescein-β-amyloid1-42 (FL-hAβ42) was purchased from rPeptide (Bogart, GA, USA). [N-ε (4-nitrobenzofurazan-7-yl)-D-Lys8]-cyclosporine A (NBD-CSA) was custom-synthesized by R. Wenger (Basel, Switzerland) [32 (link)]. PSC833 was a gift from Novartis (Basel, Switzerland). Bortezomib was obtained from Selleckchem (Houston, TX, USA). Cyclosporin A was purchased from Tocris Bioscience (Bristol, United Kingdom). Dulbecco’s phosphate-buffered saline (DPBS) with 0.9 mM Ca²⁺ and 0.5 mM Mg²⁺ was from HyClone (Logan, UT, USA). CelLytic™ M, Ficoll® PM 400, bovine serum albumin, and all other chemicals were from Sigma (St. Louis, MO, USA).