Quantifying BBB Integrity via IgG Extravasation

The BBB integrity was considered by measuring the parenchymal abundance of, IgG using semi-quantitative confocal immunomicroscopy as described previously [34 (link)-36 (link)]. Briefly, after blocking with 10% goat serum, 20 μm cryosections were incubated with goat anti-mouse IgG conjugated with Alexa488 (1:50, LifeTechnologies) for 20 h at 4°C. The sections were counterstained with DAPI. A minimum of eight and five 3-D images were captured randomly from the cortex and hippocampal formation region of the brain section, respectively with UltraVIEW Vox spinning disc confocal microscope (PerkinElmer). Total image area captured and quantified represented approximately 60% of the hippocampal formation and cortex. The voxel intensity of fluorescence of each 3-D image was analyzed with Volocity imaging software (PerkinElmer), and averaged within each region by using all eight or five 3-D images to estimate the representative voxel intensity per region per mouse. Then the mean voxel intensity of IgG extravasation was calculated within each treatment group (n = 6). The parenchymal staining of IgG was specifically selected and staining within the blood vessels were excluded based on pre-set threshold parameters of Volocity and thereafter confirmed for each image to ensure proper selection by identifying the nucleus of BBB endothelial cells.