Rotein concentration was determined with a Micro BCA Protein Assay Kit (Pierce). The tissue samples were separated by SDS-PAGE and electroblotted onto PVDF membranes. After blocking by 5 skim milk in 10 mM Tris-buffered saline (pH 7.4) containing 0.1 Tween-20 (T-TBS), the membranes were incubated with T-TBS containing the primary antibodies Tetracosactide overnight at 4uC. The membranes were then incubated with HRP-labeled secondary antibody solution (1:5,000, donkey anti-rabbit antibody; 1:20,000, sheep anti-mouse antibody, GE Healthcare) for 1 hr. The immunoreaction was visualized with an ECL chemiluminescence detection system (ECL plus or ECL prime, GE Healthcare) and digitalized by a CCD imager (LAS4000, FUJIFILM). Blot densities were quantified using the ImageJ software (Wayne Rasband, NIH, USA).AntibodiesThe primary 25033180 antibodies that we used in this study are listed in Table 1. We used anti-CB1 antibodies generated from rabbit and goat. The specificities of these antibodies were previously demonstrated by the detection of single protein band at 52 KDa, which was abolished by preadsorption with the antigen protein, in a western blot analysis of a sample of mouse telencephalon [21,22]. We also confirmed the disappearance of the immunoreactivity of CB1 in V1 by preadsorption with the antigen protein.ImmunohistochemistryFor immunohistochemistry, animals were euthanized with an overdose of isoflurane and transcardially perfused with cold PBS followed by 4 paraformaldehyde in 0.1 PB. Brains were removed from the skull and postfixed in 4 paraformaldehyde and 20 sucrose in PB overnight at 4uC. After postfixation, frozen coronal sections (30 mm in thickness) were prepared with a microtome. All immunohistochemical procedures were performed in a free-floating state. For immunoperoxidase methods, sections were washed in PBS and incubated in a mixture of 0.5 H2O2, 0.5 JW 74 site Triton X-100 in PBS for 15 min at room temperature to block endogenous peroxidase activity. Then, the sections were incubated in a blocking solution (5 normal goat or rabbit serum (Vector Laboratories), 5 bovine serum albumin (BSA) (SIGMA), 0.5 Triton X-100 in PBS) at room temperature for 4? hr. The sections were reacted with the primary antibodies in the blocking solution 
overnight at 4uC. After washing in PBS, the sections were incubated in theWestern Blot AnalysisFor western blot analysis, animals were euthanized with an overdose of isoflurane and transcardially perfused with cold 20 mM phosphate-buffered saline (PBS, pH 7.4). Brain tissue was collected immediately and frozen in powdered dry ice. Brains were sliced into 500 mm thickness by a microtome (SM 2000R, Leica Table 1. Primary antibodies used in this study.Primary antibody CB1 CB1 MAP2 Synaptophysin VGAT VGluT1 VGluT2 GAPDHImmunogen Mouse CB1, C-terminal 31 aa (443?73, NM007726) Mouse CB1, C-terminal 31 aa (443?73, NM007726) Rat brain microtubule associated proteins (MAPs) Vesicular fraction of bovine brain Mouse VGAT, 31?12 aa (BC052020) Mouse VGluT1, C-terminal 531?60 aa (NM20309) Mouse VGluT2, C-terminal 550?82 aa (BC038375)Manufacturer, catalog No., speciesConcentration/DilutionFrontier Institute, CB1-Go-Af450, goat polyclonal (Fukudome2 mg/ml et al., 2004) Frontier Institute, CB1-Rb-Af380, rabbit polyclonal (Uchigashima et al., 2007), SIGMA, M4403, mouse monoclonal Millipore, MAB5258, mouse monoclonal Frontier Institute, VGAT-Rb-Af500, rabbit polyclonal (Fukudome et al., 2004), Frontier Institute, VGluT1-Rb-Af500, rabbi.Rotein concentration was determined with a Micro BCA Protein Assay Kit (Pierce). The tissue samples were separated by SDS-PAGE and electroblotted onto PVDF membranes. After blocking by 5 skim milk in 10 mM Tris-buffered saline (pH 7.4) containing 0.1 Tween-20 (T-TBS), the membranes were incubated with T-TBS containing the primary antibodies overnight at 4uC. The membranes were then incubated with HRP-labeled secondary antibody solution (1:5,000, donkey anti-rabbit antibody; 1:20,000, sheep anti-mouse antibody, GE Healthcare) for 1 hr. The immunoreaction was visualized with an ECL chemiluminescence detection system (ECL plus or ECL prime, GE Healthcare) and digitalized by a CCD imager (LAS4000, FUJIFILM). Blot densities were quantified using the ImageJ software (Wayne Rasband, NIH, USA).AntibodiesThe primary 25033180 antibodies that we used in this study are listed in Table 1. We used anti-CB1 antibodies generated from rabbit and goat. The specificities of these antibodies were previously demonstrated by the detection of single protein band at 52 KDa, which was abolished by preadsorption with the antigen protein, in a western blot analysis of a sample of mouse telencephalon [21,22]. We also confirmed the disappearance of the immunoreactivity of CB1 in V1 by preadsorption with the antigen protein.ImmunohistochemistryFor immunohistochemistry, animals were euthanized with an overdose of isoflurane and transcardially perfused with cold PBS followed by 4 paraformaldehyde in 0.1 PB. Brains were removed from the skull and postfixed in 4 paraformaldehyde and 20 sucrose in PB overnight at 4uC. After postfixation, frozen coronal sections (30 mm in thickness) were prepared with a microtome. All immunohistochemical procedures were performed in a free-floating state. For immunoperoxidase methods, sections were washed in PBS and incubated in a mixture of 0.5 H2O2, 0.5 Triton X-100 in PBS for 15 min at room temperature to block endogenous peroxidase activity. Then, the sections were incubated in a blocking solution (5 normal goat or rabbit serum (Vector Laboratories), 5 bovine serum albumin (BSA) (SIGMA), 0.5 Triton X-100 in PBS) at room temperature for 4? hr. The sections were reacted with the primary antibodies in the blocking solution overnight at 4uC. After washing in PBS, the sections were incubated in theWestern Blot AnalysisFor western blot analysis, animals were euthanized with an overdose of isoflurane and transcardially perfused with cold 20 mM phosphate-buffered saline (PBS, pH 7.4). Brain tissue was collected immediately and frozen in powdered dry ice. Brains were sliced into 500 mm thickness by a microtome (SM 2000R, Leica Table 1. Primary antibodies used in this study.Primary antibody CB1 CB1 MAP2 Synaptophysin VGAT VGluT1 VGluT2 GAPDHImmunogen Mouse CB1, C-terminal 31 aa (443?73, NM007726) Mouse CB1, C-terminal 31 aa (443?73, NM007726) Rat brain microtubule associated proteins (MAPs) Vesicular fraction of bovine brain Mouse VGAT, 31?12 aa (BC052020) Mouse VGluT1, C-terminal 531?60 aa (NM20309) Mouse VGluT2, C-terminal 550?82 aa (BC038375)Manufacturer, catalog No., speciesConcentration/DilutionFrontier Institute, CB1-Go-Af450, goat polyclonal (Fukudome2 mg/ml et al., 2004) Frontier Institute, CB1-Rb-Af380, rabbit polyclonal (Uchigashima et al., 2007), SIGMA, M4403, mouse monoclonal Millipore, MAB5258, mouse monoclonal Frontier Institute, VGAT-Rb-Af500, rabbit polyclonal (Fukudome et al., 2004), Frontier Institute, VGluT1-Rb-Af500, rabbi.
