Frappa module

Organotypic brain slices were obtained from adult endogenous MeCP2-GFP chimeric mice, based on a slight modification of a previously described method³⁴ . Following decapitation, the brain was extracted and placed into ice-cold dissection medium composed of hibernate A (BrainBits HA), 2% B27 supplement (Gibco 17504), 2 mM L-glutamine (Gibco 25030), and 1% penicillin/streptomycin (Gibco 15140). The cerebellum and midbrain were removed and the remaining cerebral hemispheres were separated and sliced coronally at 250 μm thickness using a McIlwain tissue chopper (Ted Pella MTC/2E). The slices were gently separated from each other in chilled dissection medium and transferred onto glass-bottom dishes in culture medium containing Neurobasal A (Gibco 10888022) with 2% B27 supplement, 2 mM L-glutamine, and 1% penicillin/streptomycin. Imaging was performed immediately after brain slice preparation. FRAP experiment was performed using the Andor Revolution spinning disk confocal with the FRAPPA module (Andor Technology). Bleaching was performed using 5-7 pulses of 20 micro-second dwell time and images were collected every second. Fluorescence intensity was measured using FIJI/ImageJ v. 2.0.0-rc-65 and analyzed as described above. Post-bleach image taken 12 seconds post-photobleaching.