Rabbit anti mecp2 antibody

Animals were anaesthetized with a mix of ketamine 37.6 mg ml⁻¹, xylazine 1.92 mg ml⁻¹ and acepromazine 0.38 mg ml⁻¹, and transcardially perfused with 20 ml PBS followed by 100 ml of cold PBS-buffered 4% paraformaldehyde (PFA). The brains were removed and post-fixed overnight in 4% PFA. Next, brains were cryoprotected in 4% PFA with 30% sucrose at 4 °C for two days and embedded in Optimum Cutting Temperature (O.C.T., Tissue-Tek). Free-floating 40-µm brain sections were cut using a Leica CM3050 cryostat and collected in PBS. The sections were blocked for 1 h in 2 % normal goat serum, 0.3% Triton X-100 in PBS at room temperature. Sections were then incubated overnight at 4 °C with either rabbit anti-MeCP2 antibody (1:1,000; Cell Signaling) or rabbit anti-ASO antibody (1:10,000; IONIS Pharmaceuticals). The sections were washed three times for 10 min with PBS and incubated for 2 h at room temperature with goat anti-rabbit antibody (1:500; Alexa Fluor 488, Invitrogen, A-11034). Sections were washed again three times for 10 min with PBS and mounted onto glass slides with Vectashield mounting medium with DAPI (Vector Laboratories).