Immunostaining was then performed as previously described [32 , 33 (link)]. Mice were transcardially perfused with 4% paraformaldehyde (PFA), postfixed at 4 °C overnight, cryoprotected in 30% sucrose, and embedded in optimum cutting temperature (OCT) compound. Sections (25 μm thick) were obtained by a Leica cryostat (CM 3050S). Immunostaining was performed as previously described. Rabbit anti-calretinin (Millipore, AB5054, 1:1000), mouse anti-RFP (Abcam, ab125244, 1:500) and mouse anti-vGlut2 (Synaptic System, 135311, 1:500) were used as primary antibodies, and Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes, A11008, 1:500) and Alexa Fluor 633 goat anti-mouse IgG (Molecular Probes, A21050, 1:500) were used as secondary antibodies. Before coverslips were applied, the slides were incubated with DAPI (Sigma, D9564, 1:1000) for 15 min. Pictures were captured by a confocal microscope (Olympus, FV1000).
Images of dendritic arbors were captured under a 40 × objective lens with a confocal microscope (Olympus, FV1000) in Z-stack mode (Additional file1 ) [34 (link)]. Neuron morphology was traced manually using the NeuronJ plugin in ImageJ software. Standard morphometric analysis (Sholl analysis) was conducted as described earlier. Significance was determined by a two-way repeated-measures analysis of variance (RM 2-ANOVA; genotype and circle radius as factors).
Images of dendritic arbors were captured under a 40 × objective lens with a confocal microscope (Olympus, FV1000) in Z-stack mode (Additional file