Chicken anti mcherry

Monkeys were deeply anesthetized with 0.8 g/kg thiopental sodium one day after P3. Brains were harvested and immediately immersed in 4% ice-cold PFA and incubated at 4℃ on a shaker for 3 days (72 hours). Then, the brains were incubated in 10% sucrose at 4°C on a shaker until submersion and then sequentially incubated in 20% and 30% sucrose. Brains were coronally sectioned (40 μm) in a cryostat at -20℃ (Leica, Wetzlar, Germany). Sectioned brain slices were washed three times in 1X PBS followed by blocking in 5% normal goat serum and 0.3% Triton X-100 in PBS for 2 hours. The brain sections were incubated overnight at 4℃ in Rabbit anti-MeCP2 (1:250, 07-013, Millipore, MA, USA) and Chicken anti-mCherry (1:1,000, B205402, Abcam, Cambridge, U.K.). After washing three times, the sections were incubated in Alexa Fluor 488 goat anti-rabbit IgG (1:400, A-11008, Life Technologies, CA, USA) and Alexa Fluor 594 goat anti-chicken IgG (1:500, A-11042, Life Technologies, CA, USA). Finally, the sections were washed three times and then mounted onto coverslips using a mounting medium with DAPI solution (H-1500, Vector Laboratory, CA, USA). Confocal images were taken by using a Zeiss LSM800 Confocal Microscope (Oberkochen, Germany). MECP2 immunoreactivity was analyzed using the ImageJ software from the National Institutes of Health (MD, USA).