Rabbit anti mecp2

After DIV 12, hippocampal primary neurons were fixed for 30 min with 4% paraformaldehyde in 0.1 M phosphate buffer (PB), washed with DPBS (Invitrogen) three times and then blocked with 5% normal donkey serum in 0.1% TBS-Triton (TBS-TX) buffer for 2 h at room temperature. Primary antibodies were diluted in blocking solution at 4°C using the following dilutions: 1:1000 rabbit anti-MAP2 (Millipore), 1:1000 mouse anti-MAP2 (Sigma), 1:1000 mouse anti-Synapsin-1 (Abcam), 1:500 rabbit anti-MeCP2 (Cell Signaling Technology) and 1:500 mouse anti-MeCP2 (Sigma). After incubation in primary antibodies overnight, coverslips were incubated in the appropriate secondary antibodies diluted in blocking solution for 2 h at room temperature.
The brain tissues were fixed 2 weeks after stereotaxic injection by vascular perfusion through the left ventricle of the heart with sequential delivery of 50 ml of saline and 60 ml of 4% paraformaldehyde in 0.1 M PB. Coronal brain sections (40 μm) were prepared and processed for immunostaining using the anti-DCX (Santa Cruz; 1:300) antibodies.

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.

Experiments was performed as previously described (Heckman et al., 2014 (link)). Free-floating 45 μm sagittal sections were cut on a cryostat (Leica CM3050 S), and stained overnight with primary antibody: rabbit anti-MeCP2 (Cell Signaling 3456S); mouse anti-MeCP2 (Sigma M6818), rabbit anti-CamKII (Abcam ab52476), and 2 hr with secondary antibody: goat anti-rabbit Alexa488 (Thermo Fisher Scientific A11034); goat anti-mouse Alexa555 (Thermo Fisher Scientific A-21137). DAPI (Thermo Fisher Scientific D1306) was used to stain nuclei. Sections were mounted with ProLong Gold Antifade Mountant (Thermo Fisher Scientific P36934). Images were acquired on Zeiss 710 laser-scanning confocal microscope and Leica SP8 microscope.