2-photon microscopy:

BAM were labeled with an intracerebroventricular injection of 10 μL of Alexa Fluor 647 dextran (10 000 MW, anionic, fixable, ThermoFisher Scientific, Cat# {"type":"entrez-nucleotide","attrs":{"text":"D22914","term_id":"426839","term_text":"D22914"}}D22914; 2.5 mg/mL) in PBS as described above ^{40, 42, 44}. The next day, mice were briefly anesthetized with isoflurane (1.5–2%) and injected i.v. with dihydroethidium (10 mg/kg; ThermoFisher; Cat # {"type":"entrez-nucleotide","attrs":{"text":"D11347","term_id":"1232075","term_text":"D11347"}}D11347). One hour later, mice were re-anesthetized with isoflurane (1.5–2%) and equipped with a cranial window superfused with Ringer as described in the CBF experiments. Mice were injected retro-orbitally with fluorescein dextran-conjugated dye (2.5% w/v FITC 70 kDa) diluted in sterile saline (50 μl) to visualize the vasculature ^{43, 45} and imaged under a two-photon microscope (Fluoview FVMPE, Olympus) with a solid-state laser (InSight DS+; Spectra physics) set to an 820 nm wavelength. Image stacks were acquired through Fluoview software (FV31S-SW, v.2.3.1.163, Olympus). A map of the vasculature was taken through an x5 objective (MPlan N 5 × 0.1 NA, Olympus) to identify vessels branching from pial arteries at the cortical surface that feed the barrel area. Once the blood vessels to be imaged were identified, we switched to a 25x objective (XLPlan N 25 × 1.05 NA, Olympus) to identify BAM along the penetrating blood vessels. Then, z-stack images (509 × 509 μm²; 800 × 800 pixels) were acquired at <150 μm depth from the surface of the brain. The superfusion solution was switched from normal Ringer to Ringer containing rApoE3 (10 μg/ml; Cat# A218, Leinco Technologies) or rApoE4 (10 μg/ml; Cat# A219, Leinco Technologies). This concentration as chosen to assure sufficient penetration of ApoE into the neocortex. Forty minutes later, second images were acquired. Images were analyzed using ImageJ software. DHE fluorescence intensity in dextran-positive cells was compared before and after rApoE superfusion.