Twenty-four hours after ICH, mice were perfused under deep anesthesia with 100 ml of ice-cold PBS followed by perfusion with 30 ml formalin (10%). The brains were removed and fixed in formalin at 4 °C for a minimum of 3 days. Samples were then dehydrated with 30% sucrose in PBS and sectioned with cryostat (CM3050S; Leica Microsystems) in 10 µm coronal slices. Anti-PDGFR-β antibody (1:100, Santa Cruz), anti-PDGF-D (1:100, Santa Cruz), anti-MPO (1:100, Santa Cruz), anti-Macrophages/Monocytes (1:100, Millipore), anti-Iba1 antibody (1:100, Abcam), anti-NeuN (1:100, Abcam), anti-GFAP (1:100, Abcam) were incubated separately or double staining overnight at 4 °C. It was then incubated with the appropriate fluorescence conjugated secondary antibodies (1:200, Jackson Immunoresearch, West Grove, PA). The slices were visualized underneath a fluorescence microscope (Olympus BX51, Olympus Optical Co. Ltd., Japan), and pictures were taken with MagnaFire SP 2.1B software (Olympus, Melville, NY).
Macrophages and microglia were stained with ED1 and Iba-1 stains and these two types of cells were distinguished by their morphology as previously described (Power et al., 2003 (link)). Macrophage positive cells were quantified in the perihematoma region at 24 h using 12 fields per slide.
Macrophages and microglia were stained with ED1 and Iba-1 stains and these two types of cells were distinguished by their morphology as previously described (Power et al., 2003 (link)). Macrophage positive cells were quantified in the perihematoma region at 24 h using 12 fields per slide.