Brain Immunofluorescence

Brain sections were incubated with a blocking solution (BS; 10% goat serum, PBS) for 1 hour at room temperature followed by overnight incubation of BS with CD31 primary antibody (AF3628, 1:200) from R&D Systems (USA). Sections were then washed in PBS, incubated for 1 hour in secondary antibody (Alexa Fluor 488, 1:300) and NeuroTrace (640/680; 1:150) from ThermoFisher Scientific (USA), washed in PBS, and cover slipped with aqueous mounting medium (Immu-Mount, Thermo Scientific). Regions of interest adjacent to both the lesioned and contralesional cortex were imaged using an Axio Observer Z1 with the Apotome 2 module (Carl Zeiss). Blood vessel density was calculated as the sum of the lengths of all blood vessel segments in the image divided by the imaged area. The branching point density was computed as the number of branching points divided by the image area (see Supplemental Methods).