W plan apochromat 63 1.0 objective

Following treatment, the slice cultures were examined for mitochondrial depolarization using the JC-1 dye (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; Life Technologies, Burlington, Ontario, Canada), a positively charged cationic dye that exhibits membrane potential-dependent accumulation in mitochondria indicated by a fluorescence emission shift from red (~590 nm) to green (~525 nm). Slice cultures were incubated with 15 μM of JC-1 for 45 min at 36°C. Following incubation, slices were transferred to a temperature-controlled perfusion chamber mounted on an upright microscope (Carl Zeiss MicroImaging GmbH) with a W Plan-APOCHROMAT 63×/1.0 objective and continuously perfused with Tyrode solution containing (in mM): NaCl, 137; KCl, 2.7; CaCl₂, 2.5; MgCl₂, 2; NaHCO₃, 11.6; NaH₂PO₄, 0.4; glucose, 5.6 (pH 7.4). Images were acquired at Z = 0.25 μm and averaged four times. Image stacks were deconvolved with Huygens Essentials (Scientific Volume Imaging) and quantified using Imaris (Bitplane). Mean fluorescence intensity of the image stacks were measured separately in both the green and red channels.