Ab5054

Immunofluorescence was performed as previously described [21 (link),22 (link)]. The primary antibodies and dilutions were as follows: anti-Calretinin (Millipore, AB5054, 1:500, Billerica, MA, USA); anti-Ctip2 (Abcam, ab18465, 1:2000, Cambridge, MA, USA); anti-Foxg1 (Abcam, ab18259, 1:1000, Cambridge, MA, USA); anti-GFP (Abcam, ab13970, 1:1000, Cambridge, MA, USA); anti-Lhx2 (Abcam, ab184337, 1:500, Cambridge, MA, USA); anti-P73 (Abcam, ab40658, 1:500, Cambridge, MA, USA); anti-Prox1 (Millipore, AB5475, 1:1000, Billerica, MA, USA); and anti-Reelin (Millipore, MAB5364, 1:1000, Billerica, MA, USA). The secondary antibodies used were Alexa Fluro 488 donkey anti-chicken (Jackson Lab, 703-545-155, 1:500, West Grove, PA, USA), Alexa Fluor 546 donkey anti-rabbit (Life, A10040, 1:500, Gaithersburg, MD, USA), Alexa Fluro 647 donkey anti-rabbit (Life, A31573, 1:500, Gaithersburg, MD, USA), Alexa Fluor 546 donkey anti-rat (Life, A10040, 1:500, Gaithersburg, MD, USA), CF 568 donkey anti-rat (Sigma-Aldrich, SAB4600077, 1:500, St. Louis, MO, USA), CF 633 donkey anti-rat (Sigma-Aldrich, SAB4600133, 1:500, St. Louis, MO, USA), and Alexa Fluro 647 donkey anti-mouse (Invitrogen, A21236, 1:500, Carlsbad, CA, USA).

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 file 1) [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).