Mouse anti mecp2

Rats were deeply anesthetized with a ketamine/xylazine mixture (100/10 mg/kg) and intracardially perfused with 4% paraformaldehyde as previously described [42 (link)]. Brains were cryoprotected and cut into 40 µm coronal sections. To identify MeCP2-positive cells, a previously established protocol was used [43 (link)] with slight modifications. In short, sections were permeabilized with 0.03% PBST for 60 min at room temperature and then blocked for 60 min at room temperature with 16% normal goat serum and exposed overnight at 4°C to 1:1,000 mouse anti-MeCP2 (Abcam). Next day, the sections were washed in 1× PBS, and incubated for 60 min at room temperature with 1:1,500 anti-mouse Alexa 488-coupled IgG (Sigma-Aldrich). The sections were again washed in 1× PBS, incubated with 20 µg/mL 4′,6-diamidino-2-phenylindole (DAPI), washed, and mounted on slides. Images of each hemisphere for each region of interest (mPFC: bregma + 2.7 and BLA: bregma + 2.04) were generated with a fluorescence microscope (Axio Imager M1) and the software Zen (Zeiss) under consistent exposure time. MeCP2- and DAPI-positive cells were counted separately for each hemisphere by an investigator blinded in terms of experimental conditions via ImageJ [44 (link)].

Immunostaining for MeCP2 and β-endorphin were performed as described previously [17] (link). Brain sections (20 µm thickness) were cut on a cryostat (Leica) and collected on pre-chilled glass slides. These sections were double stained for MeCP2 (1∶500) and β-endorphin (1∶200) antibodies. The primary antibodies used were mouse anti MeCP2 (Abcam, Cambridge, MA) and rabbit anti β-endorphin (Bachem, Sam Carlos, CA). Secondary antibodies used in this study were Alexa-Flour 488 donkey anti-mouse (2 mg/ml; Invitrogen, Grand Island, NY) and Alexa Flour 594 donkey anti-rabbit IgG (2 mg/ml; Invitrogen, Grand Island, NY). After staining, pictures were taken using a confocal microscope with a 20× objective (Nikon EZ-C1 3.60 build 770, Gold version; Melville, NY). Total number of β-endorphin cells as well as total number of β-endorphin cells, located on the right and left side of the third ventricle, that were positive for MeCP2 were counted. The experimenters were blind to the experimental treatment group of the section during counting.