Anti gad67

Rats were euthanized by barbiturate (Euthasol; Virbac, Fort Worth, TX) overdose. After deep anesthesia was confirmed, cardiac perfusion was performed with saline (0.9% [w/v] NaCl solution) followed by 4% paraformaldehyde in saline. The brain was removed and post-fixed overnight in the same solution at 4°C and then placed in 30% (w/v) sucrose solution in PBS for cryoprotection. Coronal sections were cut at 40 μm thickness with a cryostat. For immunostaining, floating sections were blocked and permeabilized in PBS containing 4% normal donkey serum (NDS) and 0.5% Triton-X100 for 30 minutes on a shaker at room temperature. Sections were then incubated in PBS containing 2% NDS and mouse antibodies against glutamate decarboxylase 67 Kd isoform (anti-GAD67; Millipore cat. #: 5406; RRID: AB_2278725), a marker of GABAergic neurons overnight on a shaker at 4°C. Sections were then rinsed in PBS and incubated in PBS containing 2% NDS and AlexaFluor 594-conjugated donkey anti-mouse IgG secondary antibodies (Invitrogen cat. #: A21203; RRDI: AB_141633). After several rinses, sections were mounted with antifade mounting medium (Vectashield with DAPI; Vector Laboratory, Burlingame, CA, USA), and covered with #1.5 glass coverslips for fluorescence microscopy using an inverted microscope (Leica).

The following antibodies were used in this study: anti-GFAP (1:2000, product # G3893, Sigma, St. Louis, MO, USA) to characterize astrocytes, anti-NeuN (1:500, product # MAB377, Chemicon, Temecula, CA) to label neurons, anti-Olig2 (1:500, product # AB9610, Millipore, Billerica, MA, USA) oligodendrocyte lineage marker, anti-GAD67 (1:300, MAB5406, Millipore, Billerica, MA, USA) to label GABAergic neurons, anti-Nestin (1:300, product # 556309, BD Bioscience, San Jose, CA, USA) to label neural progenitor cells and granule cells were identified by anti-Prox-1 (1:1000, product # AB5475, Chemicon, Temecula, CA).