Immunofluorescence Staining of Mouse Brain

Immunofluorescence staining was performed on frozen tissues, as described previously (Jiang et al., 2017 (link)). In short, mouse brains were fixed by transcardial perfusion with 4% paraformaldehyde, and stored at −80°C. Frozen brain sections (35 μm thick) were obtained using a Leica cryostat, and immunostained using a free-floating staining method with the following primary antibodies: rabbit anti-GFAP (glial fibrillary acidic protein; 1:500; Millipore, Burlington, MA, USA) and rabbit anti-Iba1 (ionized calcium-binding adaptor molecule 1; 1:500; Wako, Richmond, VA, USA). Images were captured on a Zeiss Axio Imager Z2 motorized fluorescence microscope (Carl Zeiss MicroImaging). The striatal areas of the ipsilateral and contralateral hemispheres were measured and calculated as the percentage of ipsilateral/contralateral striatal area. GFAP⁺ astrocytes, Iba1⁺ microglia, and lectin-stained vessels were analyzed in a blinded fashion by measuring fluorescence intensity in four defined regions of interest (ROI) in the ipsilateral or contralateral cortex and striatum (size: 500 × 500 μm; ROI centered 1.75 mm lateral to midline/0.75 mm, 2.5 mm, 3.5 mm, and 4.0 mm below brain surface). The mean values of GFAP and Iba1 fluorescence intensity and vascular density were calculated as the percentage of ipsilateral/contralateral ROI.