Nih image j

Frozen coronal sections (40 μm) were taken through the entire brain using a Microm HM 450 sliding microtome (Thermo Fisher Scientific, Walthman, MA). Primary antibodies included anti-B220 (biotinylated, 1:500; BD Biosciences, Franklin Lakes, NJ), anti-CD68 (1:1000; AbD Serotec, Raleigh, NC), and anti-CD3e (the ε chain of the T cell receptor associated CD3 complex, 1:500; BD Biosciences). Briefly, free floating sections were immunolabeled with antibodies in conjuction with ABC Vector Elite and 3,3’-diaminobenzidine kits for visualization. B220+ and CD3e + cells were counted using the NIH Image J (Media Cybernetics, Rockville, MD) cell count function in the stroke core in 2 sections (bregma −0.46 mm and −1.94 mm) per mouse, and 3 non-overlapping fields (112 μm × 85 μm using a 20× objective). Due to the denseness and intensity of CD68 immunoreactivity within each lesion, CD68 was quantified by measuring the percent area occupied by CD68 (n = 5–8 mice per group for CD3e-immunostaining, n = 4–12 mice per group for B220-immunostaining, and n = 4–7 mice per group for CD68-immunostaining).