Vibratome

Embryos and brains dissected from newborn pups were rinsed three times in PBT (PBS/0.1% Tween-20), followed by fixation by immersion in 4% paraformaldehyde in PBT and rinsing and storage in PBT/05% sodium azide. The fixed tissues were sectioned coronally (80-100 μM) using a vibratome (Leica Microsystems).

Mice were anesthetized (Avertin, Sigma-Aldrich, St. Louis, MO) and perfused transcardially with 4% paraformaldehyde at 2 months of life. Brains were removed from the skull and post-fixed overnight at 4 C. Coronal sections (60 and 250 µm for immunofluorescence and neuronal reconstruction, respectively) were cut on a vibratome (Leica Microsystems, Mannheim, Germany) in 0.1 M phosphate buffer. For immunofluorescence brain sections were washed in phosphate-buffered saline 0.5% Triton-X and transferred in a 15 mM sodium citrate solution, pH 8.0 for 30 min at 80 C, then washed in phosphate-buffered saline with 0.5% Triton-X and blocked with blocking buffer (2% BSA in phosphate-buffered saline, 100 mm glycine, 1% Triton-X), incubated with primary antibodies overnight at 4 C (rabbit anti-Cdkl5, 1∶250, Sigma-Aldrich; mouse anti-SC35, 1∶20, rabbit anti-MeCP2, 1∶1000, Cell Signaling, Danvers, MA), incubated with an appropriate Alexa Fluor secondary antibody (1 h at room temperature), stained with DAPI, and mounted in Moviol (Calbiochem, Nottingham, UK). Images were acquired on a confocal microscope (TCS SP5 AOBS, Leica Microsystems). For neuronal reconstruction, brain sections were washed in phosphate-buffered saline, stained with DAPI, and mounted in Moviol (Calbiochem). Total dendritic length and Sholl analysis was measured using ImageJ software from confocal images.

Serial coronal sections (∼70 μm) of the brain were prepared using a Vibratome (Leica Microsystems) and processed for immunohistochemistry. For three-dimensional (3D) reconstruction, each section was analysed in sequential order from rostral to caudal using Neurolucida and StereoInvestigator (MicroBrightField). Every labelled cell in the neocortex was marked. The distribution of the nearest neighbour distance (NND) reflects the spatial point pattern of the data set, as described previously74 . Specifically, given N cells in a data set, for each cell i the distance to its closest neighbour was measured and denoted as d_i, the NND for cell i. The indicator function f (y, d) was then calculated as:

Thus, the cumulative distribution function of NND is:

Wild type, ephrin-A2 KO mice, ephrin-A5 KO mice, and ephrin-A2/A5 DKO mice were used at P10-P14 for analysis of contralateral targeting. Wild type and ephrin-A2/A5 DKO mice were used at P10-12 for analysis of topographic mapping. Mice were perfused transcardially with 0.9 % saline then 4 % PFA in PBS. Brainstems and cerebellum were extracted and post-fixed in 4 % PFA in PBS at 4 °C for 24–72 h. The cerebellum was dissected away and a small piece (100–200 μm²) of the lipophilic NeuroVue Red dye (Polysciences) was then placed in VCN on one side as described previously [8 (link), 39 (link)]. For studies of topography, a smaller piece of NeuroVue Red dye was placed on one side of either dorsal or ventral portion of VCN. Brainstems were then returned to 4 % PFA in PBS and incubated at 37 °C for 2 weeks to allow for dye to transport along the axon and into the calyces on MNTB. Brainstems were placed in 4 % low-melting agarose in PBS after incubation and sectioned coronally at 100 μm on a vibratome (Leica Microsystems). Sectioned tissue was then mounted onto chrome-alum-subbed slides and coverslipped.