Images were obtained from three sections per mouse for IHC and IF. For IHC, slides were scanned on the NanoZoomer 2.0-HT system (Hamamatsu Photonics). Images were further processed using NDP-viewing software (Hamamatsu) and Fiji software version 1.51 (National Institutes of Health). For IF images, six z-stacks per mouse were acquired on a Leica Stellaris confocal (5 or 8) with a 20x objective and 1,024 × 1,024 resolution. Quantification of confocal images were performed on a semi-automated platform using MATLAB and Imaris 9.7.1 software to create surfaces of each stain based on a threshold applied to all images and colocalize surfaces to evaluate the volume of CD206 cells within CD31+ vessels.
Quantification of confocal images for AQP4 depolarization from CD31+ vessels was performed on a semi-automated platform using MATLAB and Imaris 9.7.1 software (Bitplane) to create surfaces of each stain based on a threshold applied to all images, dilate CD31 surfaces to 2 µm, and analyses AQP4 volume outside the CD31-dilated surface.
For brain tracer-coverage measurements, six sections per mouse were used and manually thresholded to match the observed signals. All six images were quantified as area of signal/total area (DAPI coverage) and averaged to get one value per mouse.
Quantification of confocal images for AQP4 depolarization from CD31+ vessels was performed on a semi-automated platform using MATLAB and Imaris 9.7.1 software (Bitplane) to create surfaces of each stain based on a threshold applied to all images, dilate CD31 surfaces to 2 µm, and analyses AQP4 volume outside the CD31-dilated surface.
For brain tracer-coverage measurements, six sections per mouse were used and manually thresholded to match the observed signals. All six images were quantified as area of signal/total area (DAPI coverage) and averaged to get one value per mouse.