Microvascular Labeling and Immune Cell Imaging

Mice were intracardially perfused with 150 µg of Texas Red-tomato lectin (Vector Labs) at a concentration of 1 mg/ml [22] (link) at sacrifice 7 and 14 days after ICH. Mice were then perfused with 20 mL PBS followed by 20 mL 4% paraformaldehyde. Brains were removed and postfixed in 4% paraformaldehyde for 4 hours. Brains were submersed in 30% sucrose for 48 hours prior to being embedded in OCT and cut into 8 µm sections. Sections were blocked for 1 hour in 2% goat serum and stained for Ly6B.2-biotin (clone 7/4 1∶100; AbD Serotec) with a secondary streptavidin in Alexa Fluor 647 (1∶1000; Life Technologies) and Dapi. To control for unspecific staining Ly6B.2 staining, sections were stained with only secondary antibodies. Photographs were taken on an Axiovert 200M microscope (Zeiss) using the Axiovision LE program provided by Zeiss. Adjustments were made using Zen lite software.