Double Immunofluorescence Staining

All sections were first blocked with 10% normal serum blocking solution species the same as the secondary antibody, containing 3% (w/v) BSA and 0.1% Triton X-100 and 0.05% Tween 20 2 h at room temperature in order to avoid unspecific staining. The sections were then incubated with different markers as follows: NeuN (neuron marker, 1:200; Abcam), glial fibrillary acidic protein (GFAP) (astrocyte marker, 1:200; Abcam), and CD11b (microcyte marker, 1:200; Abcam). Briefly, sections were incubated with both primary antibodies overnight at 4°C, followed by a mixture of fluorescein isothiocyanate-conjugated secondary antibodies for 2 h at 4°C. The stained sections were examined with a Leica spectral confocal microscope (Germany).