Brain sections were incubated with 3% H2O2 for 15 min to block endogenous peroxidases, then washed three times with PBS and incubated for 1 h at room temperature with a blocking solution (10% goat serum). Subsequently, sections were incubated overnight with primary antibodies (GFAP, 1:500, Abcam, Cambridge, MA, USA; IBA-1, 1:200, Abcam, Cambridge, MA, USA). Then the sections were washed with PBS followed by incubation for 1 h at room temperature with biotinylated secondary antibodies. After washing with PBS again, sections were incubated for 30 min with avidin/biotinylated horseradish peroxidase. Finally, sections were washed with PBS and reacted with DAB as a chromogen.

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