Acta Neuropathol (2011) 122:293–311 Vascular corrosion casts and scanning electron microscopy All procedures were carried out as described previously [34]. In brief, anaesthetised mice (100 mg/kg pentobarbital) were ﬁrst perfused with artiﬁcial cerebrospinal ﬂuid (aCSF) con- taining heparin followed by a perfusion with 4% PFA in PBS. Subsequently, mice were perfused with the casting resin PU4ii (vasQtec, Switzerland). After a curing period of 7 days, soft tissue was macerated in 7.5% potassium hydroxide at 50 C for 24–72 h and the bones decalciﬁed in 5% formic acid at 50 C for 24–72 h. Vascular corrosion casts were washed with water, dried by lyophilisation and sputter-coated with gold particles for scanning electron microscopy (SEM) purposes. Extraction of membrane brain proteins Working on ice, brain materials (hippocampi and cortices) were homogenised separately in homogenisation buffer [Tris-HCl 10 mM, pH 7.4, EDTA 1.0 mM, sucrose 250 mM and protease inhibitors (Complete , Roche, Switzerland)] with 30 strokes using a glass Dounce homogeniser with Teﬂon pestle (Wheaton, USA). Homogenates were centri- fuged at 9009g and 4 C for 10 min. The formed supernatants were subsequently centrifuged at 110,0009g and 4 C for 75 min. Supernatants were discarded and the pellets dis- solved in solubilisation buffer [Tris-HCl 10 mM, pH 7.4, EDTA 1.0 mM, Triton X-100 0.50%, sodium deoxycholate 0.50% and protease inhibitors (Complete , Roche, Switzer- land)] by continuous rotation at 4 C for 1 h. The dissolved pellets were centrifuged at 14,0009g and 4 C for 10 min. Supernatants were collected and protein concentrations measured using the Pierce BCA Protein Assay Kit (Thermo Scientiﬁc, Rockford, IL, USA). Immunoblotting Equal amounts of total protein or equal volumes of cell cul- ture media were subjected to separation on 10–20% Tricine gels (Invitrogen, Basel, Switzerland), blotted on nitrocellu- lose membranes Immobilon-P PVDF membranes (0.45 lm, Millipore, Switzerland). The immunoblot was then incubated with primary antibodies followed by incubation with HRP- tagged secondary antibodies. Detection was performed using chemiluminescence visualised using ECL WB reagents (Amersham Pharmacia, GE, Germany) or SuperSignal West Dura Extended Duration reagents (Pierce, Rockford, IL, USA) on BIOMAX ﬁlms (GE, Germany). Immunohistochemistry Paraformaldehyde-ﬁxed and cryoprotected brains were cut into 30 lm thick slices at *-80 C using a microtome (Leica 295 Jung HN40) and kept at -20 C in an anti-freeze solution (phosphate buffer 0.50 M in MilliQ water:ethylenegly- col:glycerol = 1.3:1:1) until staining was performed. All immunohistochemical stainings were executed using the free-ﬂoating method. Washing steps were carried out between all incubations using washing buffer (TBS pH 7.4 containing 0.2% Triton X-100) at RT. Antigen retrieval was performed when required using the proteinase K antigen retrieval method [incubation of sections in proteinase K solution (proteinase K 20 lg/ml in Tris base 50 mM ? EDTA 1.0 mM, pH 8.0)] at 37 C for 7 min. Slices were blocked for 1 h at RT using blocking buffer (5.0% goat serum 5.0% donkey serum in washing buffer). Blocked slices were incubated overnight at 4 C with slight agitation in primary antibody incubation buffer (2.5% goat serum and 2.5% donkey serum in washing buffer) containing the primary antibody/antibodies. Subsequently, secondary antibody incubations were carried out for 2 h at RT. Slices were washed in washing buffer, mounted on chrom-gelatin-coated microscopy slides (SuperFrost Plus, Menzel, Braunschweig, Germany) and glass-covered using Hydromount (National Diagnostics, Hull, UK). Image analysis Fluorescent immunohistochemical images were acquired on a Leica DM4000B microscope using an Olympus DP71 colour digital camera and newCAST software (Visiop- harm, Copenhagen, Denmark). Image analysis was carried out with ImageJ software (NIH, USA). High-resolution imaging was performed using a TCS/SP2 Leica confocal laser scanning microscope (Leica, Wetzlar, Germany) with 639 objective (water, NA: 1.2) where men- tioned in the ﬁgure legends. All confocal images are maximal intensity projections of z stacks composed of multiple images. Trypan Blue BBB leakage experiments Mice received an intraperitoneal injection of 200 ll of a 0.4% Trypan Blue solution in 0.85% saline (Gibco, Swit- zerland). Thirty minutes after Trypan Blue administration, mice were perfused and their brains processed for histo- logical analysis as described above. Trypan Blue was visualised using immunoﬂuorescence with excitation and emission wavelengths at 642 and 660 nm, respectively. Protocol adapted from Persson et al. [39]. Prussian Blue and Thioﬂavin S stain for detection of haemorrhages and CAA Haemorrhages were visualised using the Prussian Blue stain method on free-ﬂoating brain sections. Free-ﬂoating brain sections were incubated in a mixture of equal 123