Immunostaining of MeCP2 and β-Endorphin in Brain

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.