780 confocal multiphoton microscope system

Imaging was performed using a Zeiss 780 confocal/multiphoton microscope system at the UVA Keck Imaging Center with Zeiss Zen software for image acquisition (Carl Zeiss, Oberkochen, Germany). For imaging cultured neurons, the excitation wavelengths used to visualize blue NPs and MitoTracker Red FM were 405 nm and 595 nm, respectively. Emission filters ranged from 410 to 500 nm for blue, and 600 nm to 700 nm for red. Magnifications of 40× or higher were used in regions of interest. For imaging live slices, the brain slices were anchored in a closed bath chamber and perfused with oxygenated sucrose-ACSF. A 405 nm laser was used as the excitation source, and emission ranged from 410 to 500 nm to capture fluorescence and 500 nm to 650 nm to capture phosphorescence. For high K⁺ measurements, the K⁺ concentration was increased from 2 to 5 mM in the perfusion solution (56.5 NaCl, 5 KCl, 5 MgSO₄, 25 NaHCO₃, 1 KH₂PO₄, 0.5 CaCl₂, 10 glucose, 107 sucrose). Ratiometric imaging was processed in MATLAB by taking the ratio of fluorescence to phosphorescence.

A subset of animals that did not undergo continuous video EEG monitoring were euthanized at p30 (Fig 1B). Brains from these animals were postfixed, processed, and sliced into sections for analysis of lesion size in the cortex, hippocampus, and thalamus. Briefly, immunofluorescence (IF) was performed, as previously described, on free-floating 40μm coronal sections [28 (link)]. 4’,6-diamidino-2-phenylindole (DAPI staining solution; ab228549; Abcam, Cambridge, United Kingdom) staining nuclear DNA was utilized to visualize anatomy and tissue injury in the fixed sections [28 (link)].
Imaging was performed using a Zeiss 780 confocal/multiphoton microscope system with Zeiss Zen software for image acquisition (Carl Zeiss, Oberkochen, Germany). For large whole-slice images, 10x magnification was used and, in regions of interest, 20x magnification was used. Excitation wavelengths used for AlexFluor488 was 488nm. Emission filter ranges blue and green were 358 to 461nm and 502 to 550nm, respectively. Tiled images with a Z-stack interval of 10 μm were stitched (overall 15% for 20x images) using Zen software.