AAV-TRE-YFP-labeled cells in fixed brain slices were filled with dye by methods described previously [31, 32]. Briefly, mice with injured cortices 1 day after stereotaxic injection of AAV-TRE-YFP were anesthetized by 1% pentobarbital and transcardially perfused with oxygenated Ringer’s solution (1.35 mM NaCl, 0.05 mM KCl, 0.01 mM MgCl2·6H2O, 0.013 mM Na2HPO4, 0.15 mM NaHCO3, 0.02 mM CaCl2·2H2O, 0.11 mM dextrose, 0.0085 mM xylocaine) and then with 4% PFA in phosphate-buffered saline (PBS; pH 7.4). Isolated brains were postfixed in 4% PFA in PBS for 1 h, and then sectioned into 75-μm slices by vibrating microtome. YFP-labeled astrocytes in the cerebral cortex were identified under a fluorescence microscope, and were patched and impaled with glass micropipettes (o.d., 1.00 mm; i.d., 0.58 mm; resistance, 100–400 MΩ) that had been backfilled with 10 mM Alexa Fluor 568 (Invitrogen) in 200 mM KCl. Patched astrocytes were iontophoretically injected with the dye by using 1-s pulses of negative current (0.5 Hz) for 2 min. After several cells were filled, the slices were placed in ice-cold 4% PFA overnight and then mounted under coverslips. Dye filled astrocytes were observed under a Zeiss710 confocal microscope equipped with a 40X oil-immersion objective, and Z-stack images were captured and projected to evaluate the subcellular distribution of injected dye and YFP.