Metamorph basic software

Fixed cerebral tissues were sectioned on a vibratome (VT1000P, Leica) at 100 μm thickness. Every sixth coronal section, for a total of six sections representative of the cranio-caudal axis between +1.10 AP to −2.46 AP, were mounted (Superfrost Plus Microscope Slides, Fisher Scientific) with ProLong Gold Antifade with 4’,6-diamidino-2-phenylindole (DAPI) (Invitrogen) and cover slipped (22×40 mm, Fisherfinest Premium Cover Glass, Fisher Scientific). A stereomacroscope (MVX10, Olympus) with a 0.63x objective (MV PLAPO, 0.15NA) and a 2x zoom was used to perform epifluorescence microscopy. Image acquisition was controlled with MetaMorph Basic software (Molecular Devices). A wavelength of 525 nm and a 607/40 nm BP emission filter were used for excitation and detection of OA-555, respectively. Exposure times were kept constant across all experimental groups and time points. ImageJ was used for image quantification. Regions of interest (ROI) were drawn around the ipsilateral and contralateral total hemisphere, cortex, white matter (corpus callosum and external capsule), hippocampus, and subcortex. The percent area above a pre-determined threshold level (50 AU, 8-bit depth) was measured in each ROI.

To verify that both treatments led to alterations in resting cell membrane potential, the voltage sensitive dye Bis-(1,3-dibutylbarbituric acid)-trimethine oxonol (DiBAC4(3)) was used [28 (link)-30 (link)]. Briefly, a stock solution was prepared at a concentration of 2 mM and diluted 1:4000 [28 (link)] in the media described above. Fluorescent images were obtained for each of the groups described above using an Olympus IX81 microscope (Olympus America, Chelmsford, Massachusetts) equipped with Metamorph Basic software (version 7.7.4.0, Molecular Devices, Sunnyvale, CA) and subsequently analyzed in ImageJ (v1.45s, NIH, Bethesda, MD). Fluorescence intensity ratio was measured as the cell membrane fluorescence intensity divided by the background intensity. A minimum of 149 cells per condition was measured. The ratio reflects the relative membrane potential with a larger ratio corresponding to a more depolarized membrane.