Immunolocalization was performed on 20 μm-thick cryosections of 23-day-old mouse eyes. Eyes were fixed in 4% fresh paraformaldehyde in phosphate buffered saline, followed by cryoprotection in 10%, 20%, and then 30% sucrose. Eyes were oriented and embedded in OCT Compound (Tissue-Tek, Sakura Finetek; Torrance, CA) just prior to sectioning. Sections were placed on glass microscope slides on a 32° C slide warmer for 30 minutes, then rinsed, post-fixed with methanol/acetone (1:1), and permeabilized with 1% triton-X 100. After blocking for 1 hour in 2% donkey serum, primary antibody was added for 24 h at 4° C. This was followed by incubation with the secondary antibody and nuclear staining for 1 hour at room temperature. Sections were then washed, coverslipped, and imaged. The primary antibody was a rabbit polyclonal anti-ELOVL4 at 1:200 dilution. This antibody was a gift from Dr. R.E. Anderson (University of Oklahoma Health Sciences Center, Departments of Ophthalmology and Cell Biology, Oklahoma City, OK). The secondary antibody was AlexaFluor® 488 goat anti-rabbit IgG (Invitrogen, Carlsbad, CA), applied at 1:200 for 1 hour. Nuclei were stained with 10 μg mL−1 DAPI. Imaging was performed on a Zeiss LSM-710 Meta laser confocal microscope with a 20x objective.
Lsm 710 meta laser confocal microscope
Manufactured by Zeiss
The LSM-710 Meta is a laser confocal microscope manufactured by Zeiss. It is designed to provide high-resolution imaging of biological samples. The microscope utilizes a set of laser sources to illuminate the sample, and a detector system to capture the reflected or emitted light. The core function of the LSM-710 Meta is to enable detailed, non-invasive imaging of microscopic structures within a sample.
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3 protocols using lsm 710 meta laser confocal microscope
1
Immunolocalization of ELOVL4 in Mouse Eyes
2
Immunolocalization of ELOVL4 in Mouse Eyes
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Immunolocalization was performed on 20-μm-thick cryosections of 23-day-old mouse eyes. Eyes were fixed in 4% fresh paraformaldehyde in PBS, followed by cryoprotection in 10, 20 and then 30% sucrose. Eyes were oriented and embedded in OCT Compound (Tissue-Tek, Sakura Finetek; Torrance, CA) just before sectioning. Sections were placed on glass microscope slides on a 32 °C slide warmer for 30 min, then rinsed, post-fixed with methanol/acetone (1:1) and permeabilized with 1% triton X-100. After blocking for 1 h in 2% donkey serum, primary antibody was added for 24 h at 4 °C. This was followed by incubation with the secondary antibody and nuclear staining for 1 h at room temperature. Sections were then washed, coverslipped and imaged. The primary antibody was a rabbit polyclonal anti-ELOVL4 at 1:200 dilution. This antibody was a gift from Dr R.E. Anderson (University of Oklahoma Health Sciences Center, Departments of Ophthalmology and Cell Biology, Oklahoma City, OK). The secondary antibody was AlexaFluor 488 goat anti-rabbit IgG (Invitrogen, Carlsbad, CA), applied at 1:200 for 1 h. Nuclei were stained with 10 μg ml−1 DAPI. Imaging was performed on a Zeiss LSM-710 Meta laser confocal microscope with a × 20 objective.
3
Immunofluorescence Assay for TDP-43 Localization
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For these assays, 293 T cells were transfected with or without plasmids and then fixed with 3.7% paraformaldehyde in PBS at RT for 15 min at 24 h (for GFP-TDP-43-IIPLD experiments) or 48 h post-transfection. The fixed cells were incubated with a TDP-43 or lamin A/C or His antibody at 4 °C overnight, followed by incubation with a secondary antibody conjugated with a fluorescent dye (Molecular Probes). The staining cells were gently fixed with 3.7% paraformaldehyde for 2 min before mounting. Slides were mounted using Vectashield DAPI H-1200 (Vector Laboratories). Cellular fluorescence images were obtained from a single optical section using an LSM710 META laser confocal microscope (Zeiss) or ELYRA super-resolution microscope (Zeiss)33 (link).
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