Stained fish skeletons were imaged using a Nikon SMZ18 stereomicroscope and NIS-Elements software. Adult fish were anesthetized with Tricaine mesylate (MS-222; 0.04%, Western Chemical Inc.) and photographed using a Canon EOS digital camera. Imaging of live and stained larval fish were performed using a Zeiss LSM 800 confocal microscope and ZEN Blue software or a Nikon SMZ18 stereomicroscope and NIS-Elements software for low magnification images of whole larvae. Larvae were anesthetized with MS-222 and embedded in 0.5% low-melting agarose for imaging. Confocal z-stacks were processed using ZEN blue software.
Lsm 800 confocal microscope
Manufactured by Nikon
The Nikon LSM 800 is a confocal microscope designed for high-resolution imaging. It utilizes laser scanning technology to capture detailed, optical sections of samples. The LSM 800 enables researchers to obtain precise, three-dimensional information about their specimens.
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Lab products found in correlation
Zen 2.5 blue edition, by Nikon (2 mentions)
A1r laser confocal microscope, by Nikon (2 mentions)
Smz18 stereomicroscope, by National Instruments (1 mentions)
Eos digital camera, by Canon (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Triiodothyronine t3, by Cayman Chemical (1 mentions)
Ts2 flfluorescence microscope, by Nikon (1 mentions)
Insulin, by Merck Group (1 mentions)
Dexamethasone (dex), by Cayman Chemical (1 mentions)
Indomethacin, by Cayman Chemical (1 mentions)
Insulin, by Cayman Chemical (1 mentions)
Isobutylmethylxanthine, by Cayman Chemical (1 mentions)
Nis elements advanced research software, by Nikon (1 mentions)
7 protocols using lsm 800 confocal microscope
1
Visualizing Fish Skeletal Structures
2
Confocal Microscope Image Analysis
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All images were generated using the Zeiss LSM800 confocal microscope, except from the time-lapse images, which were generated using the Nikon A1R confocal microscope. Objectives of 5-63x were used. Objective lenses were oil-immersed from 40x upwards. Images were analysed and processed using ImageJ (FIJI) and IMARIS (version 8.3.1).
3
Confocal Microscopy Image Acquisition
4
Quantitative Fluorescence Imaging Analysis
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Cells and tissues were imaged on a Zeiss LSM800 confocal microscope or a Nikon 800E epifluorescence microscope. Images were processed using Fiji software to adjust color palette, balance, and contrast; all changes were applied to the entire image. Fluorescence quantification and image segmentation were performed using CellProfiler (Carpenter et al., 2006 (link)). P63 fluorescence was quantified in ImageJ by creating a DAPI mask, applying it to the P63 channel, and measuring the fluorescence of individual cells. Data were exported and analyzed in Prism 6. Further image analysis including 3D stack reconstruction was conducted using Icy platform (de Chaumont et al., 2012 (link)). Student's t test was used to compare values with normal distributions. One- or two-way ANOVA with Tukey's method was used for multiple mean comparisons. For all statistical analyses, the number of animals or culture wells are noted in the figure legends. In all graphs, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Error bars represent the standard error of the mean.
5
Adipogenic Differentiation and PMSF Treatment
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Adipose tissues obtained from CIDEA reporter mice were processed for stromal vascular fraction isolation as previously described35 (link). For adipogenic differentiation, confluent cells were exposed to differentiation medium: growth medium (DMEM with 10% FBS and 1% penicillin–streptomycin) containing 2.5 mM of isobutylmethylxanthine (Cayman), 1 μM of dexamethasone (Cayman), 1 μg/ml of insulin (Sigma), 0.125 mM of indomethacin (Cayman), and 1 nM of triiodothyronine (T3, Cayman) for 3 days, and then exposed to maintenance medium: growth medium containing 1 μg/ml of insulin and 1 nM of T3 for 3 days. Differentiated adipocytes were treated with PMSFs (1 μM) in growth medium for 24 h. Zeiss LSM800 confocal microscope or Nikon Ts2-FL fluorescence microscope was used to detect fluorescence signals.
6
Confocal Microscopy Imaging Workflow
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Images were captured with a ZEISS LSM 800 confocal microscope (San Diego, CA) or with a Nikon A1R Confocal Laser Microscope (Hercules, CA) at a thickness of 0.3–0.5 µm. Confocal images were processed using the manufacturer’s software Zen 2.5 (Blue edition) or Nikon Imaging Software Element (Version 4.20) and with Adobe Photoshop.
7
Quantification of Retinal Neovascularization in Oxygen-Induced Retinopathy
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At P7, mice pups along with dams were kept in BioSpherix chamber for five days and exposed to 75% oxygen (P7–P12), and then returned to room air63 (link). The mice littermates of the same age continued at room air (21% oxygen), were considered as controls. At P17, the pups were euthanized, their eyes enucleated and fixed. The retinas were isolated and stained with rhodamine labeled isolectin B4. After staining, the retinas were flat mounted and observed under a Zeiss LSM 800 confocal microscope and retinal neovascularization was quantified using Nikon NIS-Elements advanced research software60 (link). Retinal neovascularization was highlighted in red, and the retinal neovascularization was calculated as fluorescence intensity of the highlighted area/total fluorescence intensity of the retina × 100. The percent avascular area was defined as total avascular area/total retina area × 100.
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