Sp 5035

After leaving the slides at room temperature for 1 h, they were washed with PBS in three 5 min cycles. Next, the slides were incubated in Animal-free R.T.U. Blocker (SP-5035, Vector Laboratories Inc.) overnight at 4°C. To identify astrocytes and microglia/macrophages, immunohistochemistry was performed using the primary antibodies Anti-GFAP (CPCA-GFAP, EnCor Biotech.) and Anti-Iba-1 (019-19741, Wako Chem.), respectively. Primary antibodies were diluted to 1:500 in Animal-free Blocker, R.T.U (SP-5035, Vector Laboratories Inc). The primary antibodies were then distributed across the slides and incubated at 4°C for 36 h (Figure 6C). Following three 10 min PBS wash cycles, slides were stained with secondary antibodies (Cy3 anti-rabbit, 711-165-152, Jackson Immuno.) (Cy5 anti-chicken, 703-175-155, Jackson Immuno.) diluted 1:500 in animal-free blocker and incubated at 4°C for 24 h. After three 10 min PBS washes the excess liquid was aspirated, and glass coverslips were mounted using Vector Shield medium with DAPI (H-1200, Vector Labs). Slides were kept in the dark at RT for 24 h before imaging. Image acquisition was performed on a Leica DMi8 microscope running LAS X Premium software. Peripheral components include a DFC9000 GT camera, EL-6000 light source (30 ms), HC PL APO 10x/0.45 objective, and Leica Y3 and Y5 filter cubes. Acquisitions were centered over the EDH (Figure 6C).