The sample collection procedure was the same as that for brain atrophy volume measurement. Thereafter, cells or brain sections were fixed in 4% PFA for 10 min, permeabilized in 0.3% Triton X-100 for 10 min, and then incubated in 10% bovine serum albumin (BSA) for 1 h at room temperature to block non-specific binding. Finally, they were incubated overnight at 4 °C with primary antibodies against the following as appropriate: GFAP (1:500; MAB3402, Millipore, Billerica, MA), BrdU (1:200; sc-56255, Santa Cruz, CA), Iba-1 (1:200; NB100-1028, Novusbio, Lieeleton), arginase (1:500; sc-271430, Santa Cruz, CA), CD206 (1:500, R&D System, AF2535), nestin (1:500, ab81462, Abcam, Cambridge, MA), and Sox2 (1:500, ab97959, Abcam, Cambridge, MA). For BrdU staining, brain sections were incubated with 2 M hydrochloric acid at 37 °C for 30 min and neutralized with 0.1 M sodium borate (pH 8.5) for 10 min after permeabilization and before blocking. After removing excess primary antibodies, the samples were incubated with fluorescence-tagged secondary antibodies at room temperature for 1 h. Excess secondary antibodies were removed, and the samples were incubated with 4, 6-diamidino-2-phenylindole (DAPI, BIOTIUM-23002, Life Technologies) for 10 min at room temperature. The samples were then imaged using laser scanning confocal microscopy (Leica, Soloms, Germany) 27 (link).
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