Lite software

The mice were fixed on the stage of a two-photon microscope (Leica, DM6000, Wetzlar, Germany). ACSF was perfused during the whole surgical procedure to moisten the cranial window. Throughout the experiment, body temperatures were kept constant at 36.8°C with a feedback-controlled heating pad (RWD Life Science Company, Shenzhen, China). 0.2 mL rhodamine B-dextran 70 kDa was injected intravenously before imaging to visualize the vasculature. A Leica NA 0.95 and a 25× magnification water immersion objective were used at an excitation wavelength of 800 nm. The cerebral vasculature was initially imaged with 512 × 512-pixel frames from the surface to a depth of 200 μm with two μm z-steps. Tracer movement was detected with dual channels. Imaging panels 100 μm below the cortical surface were selected to analyze tracer movement of the PVS with Leica Lite software. For paravascular and interstitial movement, circular regions of interest (ROI) 25 pixels in diameter were centered on the surrounding penetrating arterioles. Mean pixel intensities within these ROIs were measured at each time of taking pictures.

Cells were grown on glass coverslips, transfected, and infected as indicated above. At the relevant time point, the coverslips were washed with cold PBS, fixed in 4% paraformaldehyde in PBS for 10 min on ice, and permeabilized with PBS containing 0.2% Triton X-100 and 4% paraformaldehyde for an additional 10 min at room temperature. Aldehyde groups were quenched with 0.2 m glycine in PBS. Subsequent antibody incubations and washing steps followed standard protocols and have been described previously (48 (link)). Alexa Fluor fluorescent secondary antibodies were obtained from Molecular Probes and used at the manufacturer's recommended dilution. Coverslips were mounted on glass slides using Mowiol supplemented with DAPI. Imaging was performed on a Leica Sp5 confocal microscope using a ×63 oil objective. Image analysis was performed in the Leica Lite software (Leica Microsystems).