Universal blocking solution

Mice were perfused through the heart with PBS, pH 7.4, and then with 4% PFA at a rate of 3 ml/min. The brains were extracted, fixed with 4% PFA for 12 h at 4°C, and then placed in a 30% sucrose solution with 0.1% sodium azide solution at 4°C for 3 d. Forty-micrometer-thick frozen coronal sections were prepared using a cryomicrotome (CM 1950, Leica Biosystems) and transferred in 0.1 m PBS. The sections were incubated in −20°C methanol for 10 min, washed in PBS, and incubated in a blocking solution (10% donkey serum in universal blocking solution, 00-8120, Invitrogen) for 1 h at room temperature. Next, the sections were incubated with primary antibodies in PBS overnight at 4°C and washed in PBS for 3 × 5 min. Then, the sections were incubated with secondary antibodies for 2 h at room temperature and washed in PBS for 3 × 5 min. Nuclear counterstaining was performed with 100 ng/ml DAPI solution (1:10,000) in PBS for 10 min. The primary antibodies were rabbit anti-GFP antibodies (1:800, Millipore). Secondary antibodies conjugated with AlexaFluor-488 (1:350, Invitrogen) were used to visualize the signals. Fluorescence images were obtained using a TCS SP8 confocal microscope (Leica Microsystems) and a 20× objective lens. The images were analyzed with ImageJ and Imaris (Bitplane, RRID:SCR_007370) software.

Mice were perfused through the heart with PBS, pH 7.4, followed by 4% PFA. Brains were removed from the skull, postfixed with 4% PFA for 12 h at 4°C, and then placed in a 30% sucrose solution at 4°C for 3 d. Using a cryomicrotome (CM 1950, Leica Microsystems), 40-μm-thick frozen coronal sections were collected in 0.1 m PBS. Sections were incubated in −20°C methanol for 10 min, washed in PBS, and incubated in a blocking solution (10% donkey serum in universal blocking solution, 00–8120, Invitrogen) for 1 h at room temperature. Next, sections were incubated in primary antibodies in PBS overnight at 4°C and washed in PBS 3 × 5 min. Then the sections were incubated in secondary antibodies for 2 h at room temperature and washed in PBS 3 × 5 min. Nuclear counterstaining was performed with 100 ng/ml DAPI solution (1:10,000) in PBS for 10 min. Primary antibodies were used at the following concentration: rabbit anti-nNOS (1:800, Millipore) and mouse anti-GAD67 (1:300, Millipore). Secondary antibodies conjugated with Alexa-488 (1:350, Invitrogen) and Alexa-568 (1:350, Invitrogen) were used to visualize the signals. Fluorescent images for cell counting were taken using a TCS SP8 confocal microscope (Leica Microsystems) using 20× objective lens. Quantification was performed with ImageJ and Imaris software (Bitplane, RRID:SCR_007370).