Fluorescein lycopersicon esculentum lectin

Mice were anesthetized with isoflurane inhalation and 4% paraformaldehyde (PFA) perfusion. Brain samples were collected, frozen in dry ice, and cut into 20 µm thick sections (CM1900 Cryostat, Leica, Wetzlar, Germany). Three coronal sections (200µm apart) from each brain were immunostained with primary antibodies against glial fibrillary acidic protein (GFAP, astrocyte marker, 1:500, Invitrogen, Carlsbad, CA), MAO-B (1:100, Santa Cruz Biotechnology, Santa Cruz, CA), Claudin-5 (1:25, Invitrogen, Carlsbad, CA) and lectin (endothelial cell marker, 1:100, Vector Lab, Burlingame, CA). Following overnight incubation, the samples were incubated for 90 minutes with secondary antibodies: Alexa-594 or Alexa-488 IgG (Alexa 594 for red and Alexa 488 for green, 1:2000; Invitrogen, Carlsbad, CA). Vessels were stained by incubating sections overnight at 4 °C in 2g/ml with fluorescein lycopersicon esculentum lectin (Vector Laboratories Burlingame, CA). Negative controls were performed on sections collected from mice subjected to stroke and tibia fracture without applying any primary antibodies (refer to Supplementary Material Fig. 1). Images were taken using a fluorescent microscope (Nikon Microphoto-SA, Melville, NY) and analyzed using Image J software (National Institutes of Health, Bethesda, MD).