40 6 diamidino 2 phenylindole dapi

To stain perineuronal nets (PNNs), free-floating sections were washed in PBS 3 times and incubated for 2 h in a blocking solution (3% BSA in PBS for WFA staining) at RT. The sections were then incubated overnight at 4 C with Lectin from Wisteria floribunda (WFA 1:200, Sigma), a lectin that recognizes most N-acetylgalactosamine residues present in PNNs. After primary antibody incubation, the sections were washed in PBS followed by secondary antibody incubation with FITC-labeled streptavidin (1:200, Sangon Biotech). Each staining round was accompanied by nuclear counterstaining with 4 0 ,6-diamidino-2-phenylindole (DAPI 1:1,000, Solarbio) in PBS. All slides were mounted with anti-fading Fluoromount (Sigma, F4680) and left at 4 C in a dark chamber until imaging. The brain sections were acquired using identical parameters with fluorescent microscopy (Nikon Eclipse Ni), SC and V1b were identified according to Mouse Brain Atlas (Keith B.J. Franklin and George Paxinos, the third edition, Elsevier). 5 z sections were stacked for each image, exported as TIFF files. The number of WFA-positive neurons in the V1 binocular zone was counted by ImageJ in an area of 500 3 550 mm, and SC in an area of 850 3 700 mm. The average cell density of each mouse was compared between the experimental groups.