Cortex. Signal intensity was measured in layer II/III. Dotted lines indicate region boundaries. The gray lines represent the profiles in individual sections obtained from an animal, and the black line represents the mean of them. AU indicates arbitrary units. (E) Mean signal intensity of CB1 in each visual cortical region. The error bars indicate SEM (n = 5 animals, one-way repeated measured ANOVA, p,0.05, post hoc Tukey’s test, *: p,0.05). doi:10.1371/journal.pone.0053082.gblocking solution for 4? hr and then in a secondary antibody solution (1:200, species-specific biotinylated antibody (Vector Laboratories) in blocking solution) overnight at 4uC. They were then reacted using the conventional ABC-DAB method. All sections were mounted onto MAS-coated slides, dehydrated in an ascending series of ethanol, defatted in xylene, and coverslipped with DPX mountant (SIGMA). For immunofluorescence, sections were incubated in a blocking solution (5 donkey serum (Jackson ImmunoReseach), 5 BSA, 0.5 Triton X-100 in PBS) for 1? hr at room temperature. They were incubated in the blocking solution containing the primary antibodies overnight at 4uC. After washing in PBS, the sections were incubated in a secondary antibody solution (1:200, Alexa 488-conjugated or Alexa 568-conjugated species specific antibodies (Life Technologies) in the blocking solution) for 2? hr at room temperature. After washing, the sections were mounted on MAS-coated slides and coverslipped with Fluoromount/plus (Diagnostic Biosystems).Image AnalysisImage analyses were performed using the ImageJ software. Images for horizontal and layer profile analyses of CB1 immunoreactivity in the visual cortex were captured using a cooled CCD camera (VB-7010, Keyence). To measure the horizontal profile of CB1 immunoreactivity, regions of interest (ROIs) wereset on layer II/III across cortical areas. Signal intensity was measured in 12 images from 5 animals. To measure the layer profiles of signal intensity for CB1, ROIs (200 mm6800 mm) were set on layer II-VI of the binocular region of V1. CB1 immunoreactivities were measured in 12?0 sites from 3? animals in each age group. Layer and region boundaries were defined in neighboring Nissl- or DAPI-stained sections. For the synaptic localization analysis of CB1, images were acquired with laser confocal microscopy (TCS SP2, Leica Microsystems). Images were obtained using a 636 oil MK 8931 immersion objective lens (NA = 1.4, HCX PL APO, Leica Microsystems) and stored in 8-bit TIFF file format (2,04862,048 pixels; pixel size, 116.25 nm). The focus was set at a depth of 1? mm from the surface of sections. The pinhole size was set at 1.0 Airy unit, and scanning was averaged 8 times. For Alexa 488-labeled samples, the samples were excited by a 488 nm Ar laser, and the beam splitter was set to 505?30 nm. For Alexa Ebselen custom synthesis 568-labeled samples, the samples were excited by a 543 nm He/Ne laser, and the beam splitter was set to 580?25 nm. The laser power and the gain of the photomultiplier were set to exclude pixels with 0 or 255 intensity in the image. In the figures, the contrast of the images was adjusted for clearer demonstration. The colocalization of immunofluorescent signals between CB1 and each of synaptophysin, VGAT, VGluT1, and VGluT2 was evaluated by calculating Pearson’s correlation coefficient (CC).Regulation of CB1 Expression in Mouse VFigure 2. Synaptic localization of CB1 in V1. (A) Double immunofluorescent staining of CB1 (magenta) and MAP2 (green) in t.Cortex. Signal intensity was measured in layer II/III. Dotted lines indicate region boundaries. The gray lines represent the profiles in individual sections obtained from an animal, and the black line represents the mean of them. AU indicates arbitrary units. (E) Mean signal intensity of CB1 in each visual cortical region. The error bars indicate SEM (n = 5 animals, one-way repeated measured ANOVA, p,0.05, post hoc Tukey’s test, *: p,0.05). doi:10.1371/journal.pone.0053082.gblocking solution for 4? hr and then in a secondary antibody solution (1:200, species-specific biotinylated antibody (Vector Laboratories) in blocking solution) overnight at 4uC. They were then reacted using the conventional ABC-DAB method. All sections were mounted onto MAS-coated slides, dehydrated in an ascending series of ethanol, defatted in xylene, and coverslipped with DPX mountant (SIGMA). For immunofluorescence, sections were incubated in a blocking solution (5 donkey serum (Jackson ImmunoReseach), 5 BSA, 0.5 Triton X-100 in PBS) for 1? hr at room temperature. They were incubated in the blocking solution containing the primary antibodies overnight at 4uC. After washing in PBS, the sections were incubated in a secondary antibody solution (1:200, Alexa 488-conjugated or Alexa 568-conjugated species specific antibodies (Life Technologies) in the blocking solution) for 2? hr at room temperature. After washing, the sections were mounted on MAS-coated slides and coverslipped with Fluoromount/plus (Diagnostic Biosystems).Image AnalysisImage analyses were performed using the ImageJ software. Images for horizontal and layer profile analyses of CB1 immunoreactivity in the visual cortex were captured using a cooled CCD camera (VB-7010, Keyence). To measure the horizontal profile of CB1 immunoreactivity, regions of interest (ROIs) wereset on layer II/III across cortical areas. Signal intensity was measured in 12 images from 5 animals. To measure the layer profiles of signal intensity for CB1, ROIs (200 mm6800 mm) were set on layer II-VI of the binocular region of V1. CB1 immunoreactivities were measured in 12?0 sites from 3? animals in each age group. Layer and region boundaries were defined in neighboring Nissl- or DAPI-stained sections. For the synaptic localization analysis of CB1, images were acquired with laser confocal microscopy (TCS SP2, Leica Microsystems). Images were obtained using a 636 oil immersion objective lens (NA = 1.4, HCX PL APO, Leica Microsystems) and stored in 8-bit TIFF file format (2,04862,048 pixels; pixel size, 116.25 nm). The focus was set at a depth of 1? mm from the surface of sections. The pinhole size was set at 1.0 Airy unit, and scanning was averaged 8 times. For Alexa 488-labeled samples, the samples were excited by a 488 nm Ar laser, and the beam splitter was set to 505?30 nm. For Alexa 568-labeled samples, the samples were excited by a 543 nm He/Ne laser, and the beam splitter was set to 580?25 nm. The laser power and the gain of the photomultiplier were set to exclude pixels with 0 or 255 intensity in the image. In the figures, the contrast of the images was adjusted for clearer demonstration. The colocalization of immunofluorescent signals between CB1 and each of synaptophysin, VGAT, VGluT1, and VGluT2 was evaluated by calculating Pearson’s correlation coefficient (CC).Regulation of CB1 Expression in Mouse VFigure 2. Synaptic localization of CB1 in V1. (A) Double immunofluorescent staining of CB1 (magenta) and MAP2 (green) in t.
