Aprochromat

Multichannel fluorescence (DAPI–FITC–Texas Red filter set) images were captured using a Zeiss LSM 700 laser-scanning confocal microscope. For high-magnification images, a 63× oil-immersion Aprochromat objective (Zeiss) was used. Settings for gain, aperture, contrast, and brightness were optimized initially and held constant throughout each study so that all sections were digitized under the same conditions of illumination. Channels were imaged sequentially to eliminate bleed-through and multichannel image overlays were obtained using Adobe Photoshop version 7.0. Axiovision software was used to collect information on pixel immunoreactivity used for quantitative purposes.

Multichannel fluorescence images were captured using a Zeiss LSM 700 laser-scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) with a ×63 oil-immersion Aprochromat objective with an image size of 512 × 512 pixels, with a pinhole aperture of 1 Airy unit. Settings for gain, contrast, and brightness were optimized initially and held constant throughout each study so that all sections were digitized under the same conditions.
For colocalization studies, z stacks of the whole imaged neuron were taken (serial scans at different focal planes with a separation optimized by the software) as previously described in ref. 18 (link). For time-lapse imaging analysis, TMRM loaded cells were imaged in a time series of 100 images 10 s apart. The Fiji distribution (https://fiji.sc/) of ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to measure pixel signal intensity and colocalization studies.
For neurite outgrowth assays, treated primary cortical neuron cultures were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 for 4 min, and stained with Alexa Fluor 488 phalloidin, or alternatively with the neuron-specific anti–βIII-tubulin. These were then imaged by confocal microscopy, and ImageJ-Fiji software was used to measure the length of the longest neurite in each neuron.

Multichannel fluorescence (DAPI–FITC–Texas Red filter set) images were captured using a Zeiss LSM 700 laser-scanning confocal microscope, with a 63 x oil-immersion Aprochromat objective (Carl Zeiss). Settings for gain, aperture, contrast and brightness were optimized initially, and held constant throughout each study so that all sections were digitized under the same conditions of illumination. Channels were imaged sequentially to eliminate bleed-through and multichannel image overlays were obtained using Adobe Photoshop 7.0 (Adobe Systems). For time-lapse imaging analysis, cells were imaged every 15 min for 24 h while at 37 °C and 5% CO₂. The Axiovision software was used to collect information on pixel immunoreactivity used for quantitative purposes.