Ab177487

Frozen brain sections were dried at room temperature, and then pretreated with 0.3% H₂O₂ for 15 min to deactivate endogenous peroxidase. Sections were blocked with 3% normal sheep serum with 0.1% Tween 20 at room temperature for 2 h. Sections were then incubated in primary antibodies [rabbit anti-NeuN (1:500; Abcam, ab177487), rabbit anti-SOX2 (1:500; Millipore, ab5603), rabbit anti-TBR2 (1:500; Abcam, ab23345), mouse anti-PROX1 (1:200; Millipore, MAB5654), and rabbit anti-BLBP (1:500; Abcam, ab32423)] in blocking buffer overnight at 4 °C, followed by addition of the avidin-biotin-peroxidase complex (1:50; VECTASTAIN Elite ABC system, Vector Laboratories). Peroxidase was reacted in 3,3′-diaminobenzidine (5 mg/ml) and 0.075% H₂O₂ in Tris-HCl (pH 7.2). Sections were dehydrated in gradient ethanol (75% ethanol, 95% ethanol, 100% ethanol, and 100% ethanol, each for 5 min), and cleared twice in xylene for 5 min, then mounted in the neutral balsam.

After being anesthetized with euthatal (from Merck) (60 mg/kg), the Nrg1-reporting mice (2 months old) were transcardially perfused with PBS (2 ml/g of body weight), followed by 4% PFA in PBS. Brains were harvested, incubated in 4% PFA overnight, and dehydrated at 4 °C in two steps with 20% and 30% sucrose in PBS. Brains were frozen in OCT (catalog #14-373-65; Fisher) and sectioned into 40 µm slices on a cryostat microtome (Bosch Microm HM550) at − 20 °C. Brain slices were permeabilized with 0.3% TritonX 100 and 5% BSA in PBS and incubated with primary antibodies at 4 °C overnight. The brain slices were not treated with Triton- × 100 when staining with anti-GAD67 antibodies. After washing with PBS for three times, samples were incubated with Alexa Fluor-488 or -647 secondary antibodies (1:1000, ThermoFisher Scientific) for 1 h at room temperature. Samples were mounted with Vectashield mounting medium (Vector) and images were taken by Leica TCS SP8 confocal microscope. The following primary antibodies were used: rabbit anti-NeuN (1:500, Abcam, ab177487), rabbit anti-TH (1:250, Millipore, MAB152), mouse anti-NRGN (1:200, R&D, MAB7947), mouse anti-PV (1:500, Sigma, P3088), mouse anti-GFAP (1:250, Millipore, MAB360); rabbit anti-S100β (1:200, Abcam, Ab52642) and mouse anti-GABA (1:1000, Invitrogen, PA5-32241).

All sections (20 μm) were stored in a storage buffer (30% ethylene glycol and 0.01% polyvinylpyrrolidone phosphate-pH 7.4) and were permeabilized for 30 minutes with PBS containing 0.3% Triton X-100. The sections were then incubated in a blocking solution of PBS-T (0.1% tween-20 in PBS) with 10% normal goat serum and were then exposed to the following specific primary antibodies: NeuN (1 : 200, Abcam ab177487, United Kingdom), GFAP (1 : 200, Millipore MAB360, Billerica, MA), Iba-1 (1 : 200, Wako 019–19741, Japan), and cleaved caspase-3 (1 : 200, cell signaling technology 9661L, Beverly, MA). Sections were then incubated with Alexa fluor 488- (1 : 500, Thermo fisher A-11001 and A-11008, Waltham, MA) or Alexa fluor 594-conjugated secondary antibodies (1 : 500, Thermo fisher A-11005 and A-11037) for 2 hours in the dark. We focused on the slices locating at 1,21 and −3.60 mm from the bregma and dashed red square in the cortex indicates the photographed area (Supplementary Figure S1). The fluorescence images were captured with a Zeiss LSM 700 laser scanning confocal device (Carl Zeiss, Jena, Germany), and image analysis was performed using ImageJ software (NIH, Bethesda, MD).