Fluorescence immunohistochemistry with floating frozen sections was performed as previously described [23 (link)]. Briefly, primary antibodies were as follows: rat anti-GFAP (Invitrogen); goat anti-IBA-1 (Abcam); rat anti-Glut-1 (Abcam); rabbit anti-Olig2 (Millipore); rabbit anti-NeuN (Abcam); and rat anti-CD31 (BD Biosciences). Secondary antibodies were conjugated to Alexa Fluor 594 Streptavidin, Alexa Fluor 647 Streptavidin, Alexa Fluor 647 donkey anti-rat, and Alexa Fluor 488 donkey anti-rabbit (Jackson ImmunoResearch). For the quantification of GFAP, Glut-1, and IBA-1 immunoreactive areas, 3 fields (650 μm × 450 μm) within peri-infarct cortex were precisely taken from 3 independent tangential sections of each animal using a 40 × objective with confocal microscopy (Nikon C2). The parameters for scanning were kept constant across treatment conditions. Single images were analyzed using ImageJ by investigators blind to all treatment groups. Average percentage of the fluorescent staining area per image was calculated for each condition.
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