Bright field microscopy and visualization of DAPI- and SYBR green-stain was executed using a Leica DC490 digital camera equipped with a UV light source mounted onto a MZ-FLIII Leica dissection microscope. When appropriate, linear adjustments were made on colour contrast and brightness using the image-processing software Adobe Photoshop CS6 for Apple Macintosh (Adobe Systems Inc.).
Mz fliii dissection microscope
Manufactured by Leica
The Leica MZ-FLIII is a dissection microscope designed for a range of applications. It features a trinocular observation tube, providing the user with the option to connect a camera or other imaging device. The microscope is equipped with a 6.3:1 zoom ratio, allowing for flexible magnification adjustments. Additionally, it offers LED illumination for consistent and efficient lighting during observations.
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Lab products found in correlation
Dc490 digital camera, by Leica (2 mentions)
Dc490, by Leica (2 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
Alizarin red s, by Merck Group (1 mentions)
Acetone, by Merck Group (1 mentions)
Alizarin red, by Merck Group (1 mentions)
Alcian blue, by Merck Group (1 mentions)
Alcian blue 8gx, by Merck Group (1 mentions)
Geneart, by Thermo Fisher Scientific (1 mentions)
Tcs sp8 confocal microscope, by Leica (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Its x, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Semi solid methylcellulose, by STEMCELL (1 mentions)
Dfc300fx camera, by Leica (1 mentions)
11 protocols using mz fliii dissection microscope
1
Brightfield Microscopy and DAPI/SYBR Green Staining
2
Visualizing Embryonic Development
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Photographing of stained embryos including the detection of the nuclear dye SYBR green and thin-sectioned embryos were performed under a MZ-FLIII Leica dissection microscope equipped with a Leica DC490 digital camera and an external UV-light source. Whenever indicated, linear adjustments were performed on colour, contrast and brightness using the image-processing software Adobe Photoshop 2022. The phylogenetic trees were made using FigTree V1.4.4.
3
Embryo Imaging via Bright-field and Confocal Microscopy
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Bright-field microscopy and documentation of DAPI counterstained embryos were performed using a Leica DC490 digital camera equipped with a UV light source mounted onto a MZ-FLIII Leica dissection microscope. For confocal microscopy, an inverted Leica TCS SP5 confocal microscope was used. For the detection of Fast Red and DAPI signal, the emission wavelengths were 561 nm and 404 nm respectively, and the collected wavelengths were between 600 nm and 642 nm for Fast Red, and between 430 nm and 550 nm for DAPI.
When appropriate, contrast and brightness were adjusted using the image-processing software Adobe Photoshop CS6 for Apple Macintosh (Adobe Systems Inc.) and GIMP 2.10.0 for Linux ([43 ] Kimball et al. 2018).
When appropriate, contrast and brightness were adjusted using the image-processing software Adobe Photoshop CS6 for Apple Macintosh (Adobe Systems Inc.) and GIMP 2.10.0 for Linux ([43 ] Kimball et al. 2018).
4
Photographing Stained Embryos with Leica Microscope
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A MZ-FLIII Leica dissection microscope equipped with a Leica DC490 digital camera and an external UV-light source was used to photograph stained embryos. Whenever necessary and justified, linear adjustments were performed on color, contrast, and brightness using the image-processing software Adobe Photoshop CC 2018.
5
Embryo Imaging Using Leica Microscope
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Embryos were photographed using a Leica DC490 digital camera equipped with a UV light source mounted onto a MZ-FLIII Leica dissection microscope. Brightness, contrast, and color values were adjusted in all images using the image processing software Adobe Photoshop CC 2018 (for Apple Macintosh (Adobe Systems Inc. San Jose, CA, USA).
6
Embryo Dissection and Staining Protocol
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Staining of embryos was either documented from whole mounts, in the form of flat-mounted parts of the embryos, or in the form of dissected appendages. For the dissection of appendages, we used fine tungsten needles recycled from burned-out old-fashioned light bulbs that were sharpened in the flame of a Bunsen burner.
Bright field microscopy and visualization of the nuclear dye SYBR-green were performed under a MZ-FLIII Leica dissection microscope using a Leica DC490 digital camera equipped with an external UV-light source. Whenever necessary and appropriate, linear adjustments were performed on color, contrast and brightness with the image-processing software Adobe Photoshop CC 2018.
Bright field microscopy and visualization of the nuclear dye SYBR-green were performed under a MZ-FLIII Leica dissection microscope using a Leica DC490 digital camera equipped with an external UV-light source. Whenever necessary and appropriate, linear adjustments were performed on color, contrast and brightness with the image-processing software Adobe Photoshop CC 2018.
7
Skeletal Staining in Mice
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Newborn mice and 3–4 week old mice of both sexes were euthanized and prepped for alizarin red and alcian blue skeletal staining (Ovchinnikov, 2009 ) by fixing in 100% ethanol for 24 h. Samples were then switched to acetone (Sigma-Aldrich) for an additional 24 h. Once fixed, samples were stained with final concentration of 5% glacial acetic acid, 0.5% alizarin red S (Sigma-Aldrich), 0.9% alcian blue 8GX (Sigma-Aldrich) in ethanol for 3 h at 37 °C and then at room temperature for 24 h. Samples were then placed in 1% KOH (Amresco) for 3 h and replaced with fresh KOH until non-bone tissue was transparent. Samples were then replaced with increasing concentrations of glycerol and photographed with a Leica MZFLIII dissection microscope with Diagnostic Instruments 14.2 Color Mosaic camera for newborn samples. 3–4 week old samples were photographed with a Nikon E5200 without a microscope.
8
Stable Transformation of A. thaliana with B. napus MGAT2-GFP
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The stable transformation of A. thaliana seeds (ecotype Columbia) with B. napus napinFP1::Musmu-MGAT2-GFP was performed using the floral dip method (Clough and Bent, 1998 (link)). The coding region of the Geneart (Life Technologies, Regensburg, Germany) synthetic construct 0954364_Musmu-MGAT_pMA (NM_177448), contained within an EcoRI fragment, was inserted into a custom vector pJP3386 (Supplementary Figure S1 ) at the EcoRI site, creating pJP3390. The seeds from the transformed plant were viewed after maturation under a Leica MZFLIII dissection microscope coupled to 0.5x M-series objective, 1x C-mount adaptor, and 100-W mercury-vapor burner for fluorescence imaging (Leica Microsystems). Transgenic seeds (strongly GFP positive) and Non-transgenic (GFP negative) seeds were selected. Subsequent generations of transgenic A. thaliana plants expressing FP1::MGAT2 were grown in a random design alongside vector-only pORE04 (AY562534-AY562548; Arabidopsis Biological Resource Centre) and Columbia (parental) controls as described above.
9
Immunofluorescence Imaging of Mammary Gland
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Antibodies were diluted in PBS containing 2% BSA. Primary colonies were fixed with 4% paraformaldehyde at room temperature for 10 min. Colonies were then blocked in PBS containing 0.2% Triton X-100 and 10% goat serum for 1 h, followed by incubation with rabbit anti-phospho-Lrp6 Ser1490 antibody overnight at 4°C. The samples were washed with PBS containing 2% BSA for 3 times and incubated with secondary antibody for 1 hr. The samples were finally washed with PBS for 3 times and stained with DAPI. Whole mount fluorescent images of mammary glands were obtained using a Leica MZFLIII dissection microscope. RNA in situ images were acquired using a Zeiss A1-AXIO upright microscope. For confocal imaging, mammary glands were minced and then coverslipped. Confocal Images were acquired through a 40× or 63× oil immersion objective on a Leica TCS SP8 confocal microscope.
10
Isolation and Culture of Endothelial Cells
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Freshly isolated endothelial cells were cultured on fibronectin (Sigma)-coated 24-well plates in IMDM medium containing 15% FBS, 1% BSA, 10−4 mM 2-mercaptoethanol (Sigma), 1% Glutamax, 1% ITS-X (Insulin, transferrin and Selectin, Life Technologies), 100 ng/ml rmVEGFa (R&D systems) and 50 ng/ml rmbFGF (Life Technologies) at 37 °C in a humidified 5% CO2 atmosphere (Esco Medical). Cells were passaged using 0.05% trypsin when reaching 80%-90% confluence, fresh medium was supplied every 2-3 days depending on cell confluence. For in vitro endothelial cell colony assays, freshly isolated cells were plated in triplicate in 0.5 ml of semi-solid methylcellulose (Stem Cell Technologies), supplemented with 100 ng/ml rmVEGFa. Colonies were scored after 7 days. Passage of primary colonies was achieved by single colony picking using a sterile pipette tip followed by digestion into single-cell suspension using 0.05% trypsin. Single cells were then re-plated into freshly supplemented methylcellulose medium. Colonies containing 10 or more cells were counted. To visualize the overall density of colonies in an entire well, the culture contents were fixed with 4% PFA followed by wash and 30-min incubation with Crystal Violet dye and visualized using Leica MZ FLIII dissection microscope.
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