Confocal fluorescence-microscopy to visualize tubulin aggregates, multinucleation, Annexin V-FITC/PI or Alexa Fluor 594 TUNEL-staining in HTLV-1 Tax-expressing cells was performed on a Zeiss LSM800 instrument with an Airyscan super-resolution detector and stage CO2 incubator (Carl Zeiss Microscopy). All images were taken using either Plan-Apochromat 40x/1.3 or Plan-Apochromat 63x/1.4 oil immersion objectives and Zeiss ZEN system software. The expression of HTLV-1 p30II-GFP in cotransfected cells was visualized on an inverted Nikon Eclipse TE2000-U microscope and D-Eclipse C1 confocal system (Nikon Instruments, Melville, NY) equipped with 633 nm and 543 nm He/Ne and 488 nm Ar lasers using a Plan Apo 20x/0.75 objective lens.
D eclipse c1 confocal system
Manufactured by Nikon
The D-Eclipse C1 is a confocal microscopy system designed for imaging and analysis of biological samples. It features a modular design that allows for customization to suit various research needs. The system utilizes laser illumination and a pinhole aperture to provide optical sectioning and improved image resolution compared to conventional widefield microscopy.
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Lab products found in correlation
Lsm 800, by Zeiss (1 mentions)
Zen system software, by Zeiss (1 mentions)
Eclipse te2000 u microscope, by Nikon (1 mentions)
Plan apochromat 40x 1, by Zeiss (1 mentions)
Plan apochromat 63x 1, by Zeiss (1 mentions)
Airyscan super resolution detector, by Zeiss (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Anti hif 1α, by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Anti jnk2, by Cell Signaling Technology (1 mentions)
Eclipse microscope, by Nikon (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
Eclipse 90i microscope, by Nikon (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
4 protocols using d eclipse c1 confocal system
1
Visualizing HTLV-1 Tax-induced Cellular Stress
2
Immunofluorescent Analysis of HIF1α and JNK2
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Paraffin-embedded whole-tissue sections were sectioned at 6 μm thickness and deparaffinized. Antigen retrieval was performed by heating in 0.1 mol/l sodium citrate (pH 6.0) using a microwave. Slides were then incubated overnight at 4 °C with a mixture of two primary antibodies, anti-HIF1α (Sigma) and anti-JNK2 (Cell Signaling), using antibody diluents (IHC World). Slides were rinsed with PBS and then incubated for 1 h with Alexa Fluor 594 or Alexa Fluor 488 (Life Technologies). Slides were mounted with ProLong Gold anti-fade reagent containing 4′,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific). Cell death was analyzed using the in situ cell death detection kit (Roche), following the manufacturer’s instructions. Confocal images were obtained using a Nikon D-Eclipse C1 confocal system and processed with Nikon EZ-C1 3.90 and Adobe Photoshop software.
3
Confocal Imaging of Whole Mount Muscle
4
Immunofluorescence Imaging of Muscle Tissues
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Antibodies used were against GFP (1:500; Invitrogen, Life Technologies, Grand Island, NY, USA), quail endothelial marker Qh1 (1:10) and Pax7 (1:10) (both from Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA), desmin (1:100; MP Biomedicals, Santa Ana, California, USA) and SMA (1:200; Sigma-Aldrich, Rehovot, Israel), both recognizing smooth and striated muscle [65 (link)], and ZO-1 (1:100, Zymed, Life Technologies, Grand Island, NY, USA). Secondary antibodies were coupled either to Cy2 or Rhodamine (1:100; Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Nuclei were visualized with Hoechst.
Images were photographed using a DP70 (Olympus) cooled CCD digital camera mounted on a BX51 microscope (Olympus) [14 (link)]. Confocal sections of whole-mount preparations encompassing their entire thickness were photographed using a Nikon Eclipse 90i microscope with a Plan Apo 10x/0.45 dry objective (Nikon) and a D-Eclipse c1 confocal system (Nikon) at 2.7 μm increments through the z-axis. Images were z-stacked with EZ-C1 3.90 FreeViewer software. For figure preparation, images were exported into Photoshop CS3 (Adobe). If necessary, the levels of brightness and contrast were adjusted to the entire image and images were cropped without color correction adjustments or γ adjustments. Final figures were prepared using Photoshop CS2.
Images were photographed using a DP70 (Olympus) cooled CCD digital camera mounted on a BX51 microscope (Olympus) [14 (link)]. Confocal sections of whole-mount preparations encompassing their entire thickness were photographed using a Nikon Eclipse 90i microscope with a Plan Apo 10x/0.45 dry objective (Nikon) and a D-Eclipse c1 confocal system (Nikon) at 2.7 μm increments through the z-axis. Images were z-stacked with EZ-C1 3.90 FreeViewer software. For figure preparation, images were exported into Photoshop CS3 (Adobe). If necessary, the levels of brightness and contrast were adjusted to the entire image and images were cropped without color correction adjustments or γ adjustments. Final figures were prepared using Photoshop CS2.
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