*5.9. Brain Capillary Harvest and Western Blot Analysis*

Protein expression levels in brain capillaries were analysed by Western blotting as previously described [18,72,86]. Brain capillaries were homogenised in lysis buffer (Sigma-Aldrich, St Louis, MO, USA) containing Complete® protease inhibitor (Roche, Mannheim, Germany). Homogenised samples were centrifuged at 100,000× *g* for 90 min. Brain capillary membranes were resuspended in buffer containing protease inhibitor and stored at −80 ◦C.

Western blots were performed using the Invitrogen NuPageTM 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 (Abcam): 1:100 (1 µg/mL); β-actin: 1:1000 (1 µg/mL); LRP-1: 1:750 (1 µg/mL)). Membranes were washed and incubated for 1 h with horseradish peroxidaseconjugated ImmunoPure® secondary IgG (1:10,000; Pierce, Rockford, IL, USA). Proteins bands were detected via enhanced chemiluminescence and recorded using a BioRad Gel Doc 2000TM gel documentation system (BioRad, Hercules, CA, USA).
