Adsorption 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, 16574785 31?12 aa (BC052020) Mouse VGluT1, C-terminal 531?60 aa (NM20309) Mouse VGluT2, C-terminal 550?82 aa (BC038375)Manufacturer, catalog No., speciesConcentration/Eledoisin custom synthesis 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, rabbit polyclonal Frontier Institute, VGluT2-Rb-Af720, rabbit polyclonal 2 mg/ml for immunohistochemistry, 0.5 mg/ml for western blot 1:500 2 mg/ml 2 mg/ml 2 mg/ml 2 mg/ml 0.05 mg/mlGlyceraldehyde-3-phosphate dehydrogenase Millipore, MAB374, mouse monoclonal from rabbit muscledoi:10.1371/journal.pone.0053082.tRegulation of CB1 Expression in Mouse VFigure 1. Distribution of CB1 in the visual cortex. (A) Low-magnification image of a coronal section of mouse brain at P30, immunostained for CB1. Inset, magnified view of LGN (*). Scale, 1 mm and 250 mm (inset). (B) Layer distribution of CB1 immunoreactivity in V1 (CB1). Layer boundaries were determined in neighboring Nissl-stained sections (Nissl). Scale, 100 mm. (C) Regional distribution of CB1 immunoreactivity in the visual cortex. Arrowheads indicate the boundaries between V1 and V2, determined in Nissl-stained sections. V2M: secondary visual buy Calcitonin (salmon) cortex medial area, V2L: secondary visual cortex lateral area, MR: monocular region, BR: binocular region. Scale, 500 mm. (D) Horizontal profiles of CB1 immunoreactivity across the visual.Adsorption 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, 16574785 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, rabbit polyclonal Frontier Institute, VGluT2-Rb-Af720, rabbit polyclonal 2 mg/ml for immunohistochemistry, 0.5 mg/ml for western blot 1:500 2 mg/ml 2 mg/ml 2 mg/ml 2 mg/ml 0.05 mg/mlGlyceraldehyde-3-phosphate dehydrogenase Millipore, MAB374, mouse monoclonal from rabbit muscledoi:10.1371/journal.pone.0053082.tRegulation of CB1 Expression in Mouse VFigure 1. Distribution of CB1 in the visual cortex. (A) Low-magnification image of a coronal section of mouse brain at P30, immunostained for CB1. Inset, magnified view of LGN (*). Scale, 1 mm and 250 mm (inset). (B) Layer distribution of CB1 immunoreactivity in V1 (CB1). Layer boundaries were determined in neighboring Nissl-stained sections (Nissl). Scale, 100 mm. (C) Regional distribution of CB1 immunoreactivity in the visual cortex. Arrowheads indicate the boundaries between V1 and V2, determined in Nissl-stained sections. V2M: secondary visual cortex medial area, V2L: secondary visual cortex lateral area, MR: monocular region, BR: binocular region. Scale, 500 mm. (D) Horizontal profiles of CB1 immunoreactivity across the visual.
