Vesicles were imaged using a Nikon Eclipse confocal laser scanning microscope using a × 60 water immersion objective. All the images were collected and stored at a 512 × 512 resolution. Three different scans were performed for each vesicle: (1) excitation at 488 nm, with a 500–530-nm emission filter (donor scan); (2) excitation at 488 nm, with a 565–615-nm emission filter (FRET scan); and (3) excitation at 543 nm, with a 650-nm longpass filter (acceptor scan). Gains of 8.0 were used for all the three scans. The bleaching of the fluorescent proteins was minimized through the use of ND8 filters when exciting with the 488-nm laser, and low pixel dwell time (1.68 μs).
Eclipse laser scanning confocal microscope
Manufactured by Nikon
The Eclipse laser scanning confocal microscope is a laboratory equipment designed for high-resolution imaging. It utilizes a focused laser beam to scan samples, allowing for the collection of optical sections and the reconstruction of three-dimensional images. The core function of this microscope is to provide detailed, high-quality visual data for research and analysis purposes.
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Lab products found in correlation
Alexa fluor 488 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Permanox chamber slide, by Thermo Fisher Scientific (1 mentions)
Donkey anti rabbit cy3, by Jackson ImmunoResearch (1 mentions)
Fluoromyelin red, by Thermo Fisher Scientific (1 mentions)
Streptavidin fitc, by Thermo Fisher Scientific (1 mentions)
Antibody diluent, by Agilent Technologies (1 mentions)
Biotinylated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Normal goat serum (ngs), by Jackson ImmunoResearch (1 mentions)
Nis elements software, by Nikon (1 mentions)
5 protocols using eclipse laser scanning confocal microscope
1
Confocal Imaging of Fluorescent Vesicles
2
Imaging Bacterial Biofilms with CLSM
3
Immunofluorescence Analysis of Neuromuscular Junctions
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Gastrocnemius muscle was dissected from perfused mice and prepared as described in the immunofluorescence section. Floating 40 μm thick longitudinal sections of gastrocnemius were incubated in a blocking solution containing PBS1x, 0.5% Tween-20, 1.5% BSA for 4 hours at room temperature and then in PBS1x, 0.3% Triton X-100 overnight at room temperature with the polyclonal rabbit anti-synaptophysin antibody at 1:50 (Invitrogen). The sections were washed with PBS1x and then incubated first with donkey anti-rabbit Cy3 (Jackson ImmunoResearch) and α-bungarotoxin-Alexa488 (Invitrogen) at 1:500 for 1 hour at room temperature and then with Fluoromyelin red (Invitrogen) at 1:300 for 30 min. The sections were further washed with PBS1x and mounted. Analysis was performed on a Nikon Eclipse laser scanning confocal microscope. A total of approximately 1,000 neuromuscular junctions were counted from at least 10 sections of gastrocnemius. Individual NMJs were considered as innervated when synaptophysin staining covered at least 50% of the area of α-bungarotoxin staining.
4
Immunohistochemical Evaluation of Misfolded SOD1
5
Spinal Cord Immunohistochemistry in Mice
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Mice were perfused intracardially and fixed with 4% paraformaldehyde in 0.1 M Sorenson’s phosphate buffer, pH 7.2, the entire spinal cord was dissected, post-fixed for 2 hours in the same fixative and transferred in a 30% sucrose phosphate buffer for at least 2 days. The lumbar spinal cord or brain was embedded in OCT compound (Sakura) and snap frozen in isopentane (2-methylbutane) cooled at −40°C on dry ice. Floating lumbar spinal cord or brain cryosections (30μm or 35μm, respectively) were incubated in a blocking solution containing PBS1x, 0.5% Tween-20, 1.5% BSA for 1.5 hours at room temperature and then in PBS1x, 0.3% Triton X-100 overnight at room temperature with the primary antibodies (listed in antibodies section). Primary antibodies were washed with PBS1x and then detected using donkey anti-rabbit or anti-mouse FITC or Cy3 (1:500) coupled secondary antibodies (Jackson ImmunoResearch). The sections were washed with PBS1x and mounted. Analysis was performed on a Nikon Eclipse laser scanning confocal microscope. Fluorescence intensity from unsaturated images captured with identical confocal settings (minimum of four spinal sections were imaged per animal) was quantified using NIS elements software (Nikon).
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