Rabbit anti cdkl5

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.

The following primary antibodies were used in immunofluorescence and western blotting experiments: mouse anti-bassoon (Santa Cruz, sc-58509), rabbit anti-CDKL5 (Sigma, HPA002847), mouse anti-CDKL5 (Santa Cruz, sc-376314), rabbit anti-collybistin (SYSY, 261003), rabbit anti-GABA_AR γ₂ (SYSY, 224003), goat anti-GABA_AR γ₂ (Invitrogen, PA5-19299), rabbit anti-GAPDH (Sigma, G9545), mouse anti-gephyrin (Santa Cruz, sc-25311), anti-GFP (chicken, Molecular Probes, A10262), chicken anti-MAP2 (SYSY, 188006), mouse anti-c-myc (clone 9E10), rabbit anti-VGAT (SYSY, 131003). Secondary Alexa Fluor anti-rabbit, -mouse and -chicken were purchased from Abcam or Invitrogen. HRP-conjugated goat anti-mouse, goat anti-rabbit and donkey anti-goat secondary antibodies for western blottings were purchased from Jackson Immunoresearch.