Immunohistochemical Analysis of CD36 and IBA1 in SBI

Immunohistochemistry was performed at 24 hours after SBI as described previously [5 (link), 7 (link)]. Briefly, the deep anesthetized rats were intracardially perfused with ice-cold PBS and 10% formalin. The whole brains were removed from skull and postfixed in 10% formalin at 4°C for 24 hours followed by the dehydration in 30% sucrose in PBS. The brains were sectioned into 10 μm thick coronal slices using a cryostat (CM3050S; Leica Microsystems, Bannockburn, IL). The slices were coincubated with the primary antibodies of rabbit polyclonal anti-CD36 (1 : 100, Santa Cruz Biotechnology, Inc, Dallas, TX) with goat polyclonal anti-ionized calcium-binding adapter molecule 1 (IBA1, 1 : 200, Abcam, Cambridge, MA) overnight at 4°C, followed by corresponding FITC-conjugated and Texas Red-conjugated secondary antibodies (1 : 400, Jackson ImmunoResearch, West Grove, PA) for 2 hours at room temperature. The fluorescence staining was visualized with a fluorescence microscope (Olympus BX51).