Immunofluorescence Staining of Brain Tissue

Immunofluorescence procedures were performed as described previously [31 (link), 32 (link)]. Mice were deeply anesthetized and perfused transcardially with 30 mL of 0.9% saline solution (37 °C) followed by 100 mL of 4% paraformaldehyde (Sigma) in 0.1 mol/L phosphate buffer (pH 7.4). Subsequently, brains were removed and kept in 4% paraformaldehyde (Sigma) overnight at 4 °C and then embedded in paraffin. The brains were cut into 5-μm-thick slices, which were incubated with goat anti-Iba-1 (1:500, Abcam), rabbit anti-NeuN (1:200, Millipore), mouse anti-GFAP (1:500, Abcam), or rabbit anti-CHRNA7 (1:200, BOSTER) overnight at 4 °C. Finally, the slices were incubated with the corresponding secondary antibody for 2 h. The sections were analyzed with an Olympus FluoView 1200 confocal microscope system (Olympus Corporation, Tokyo, Japan), and photomicrographs of representative fields were taken.