Hek293T cells were plated in 24-well plates on cover glass (Carl Roth) coated with poly-L-Lysine (Sigma–Aldrich). Cells were transfected with 125 ng of plasmid, and total plasmid levels were adjusted to 500 ng with empty plasmid. For immunofluorescence analysis, cells were chemically fixed for 15 min with 4% formaldehyde (16% formaldehyde diluted in PBS, Thermo Scientific), washed 3× in PBS followed by permeabilization and blocking in PBS containing 0.1% Triton X-100 (EMD Millipore) and 20% fetal calf serum (TPBS-FCS) for 45 min. Subsequently, the fixed cells were probed with anti-TOM20 and anti-FLAG antibodies in TPBS-FCS for 1 h and washed 3× in PBS. After staining with fluorescently labeled secondary antibodies Alexa Fluor 488 and Alexa 633, both diluted in TPBS-FCS for 1 h, the slides were washed 3× in PBS, followed by Hoechst staining (1 μg/ml in PBS) for 10 min. After washing 3× with PBS, the cover glasses were mounted with Fluoromount-G (Southern Biotech) on microscope slides. Samples were imaged with a LSM780 system (Carl Zeiss) using 405, 488, and 633 nm laser lines, a Plan-APOCHROMAT 63x 1.4NA oil objective (Carl Zeiss) and pinhole settings of 1AU with the Zeiss ZEN 2010 software. Image processing was performed using ImageJ (http://rsb.info.nih.gov/ij/ ).
Plan apochromat 63x 1.4 na oil objective
Manufactured by Zeiss
Sourced in Germany
The Plan-Apochromat 63X/1.4 NA oil objective is a high-performance microscope objective designed for advanced imaging applications. It features a high numerical aperture of 1.4 and a magnification of 63X, providing excellent optical performance and resolution. The objective is designed to be used with immersion oil to optimize image quality. Its core function is to provide high-quality, detailed imaging for various scientific and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Axiovert 200m, by Zeiss (6 mentions)
Lsm 710, by Zeiss (4 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Opal 520, by Akoya Biosciences (2 mentions)
Mm per2 c1 and mm arntl c2 rna probes, by Akoya Biosciences (2 mentions)
Opal 570 dye, by Akoya Biosciences (2 mentions)
Rnascope multiplex fluorescent assay, by Advanced Cell Diagnostics (2 mentions)
Vt1200, by Leica (2 mentions)
Dapi fluoromount g, by Southern Biotech (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Cover glass, by Carl Roth (1 mentions)
Lsm780 system, by Zeiss (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
μ slide chemotaxis chamber, by Ibidi (1 mentions)
Axiovision extended focus module, by Zeiss (1 mentions)
Molecular probe, by Thermo Fisher Scientific (1 mentions)
Axiovert 200m epifluorescence microscope, by Zeiss (1 mentions)
16 protocols using plan apochromat 63x 1.4 na oil objective
1
Immunofluorescence Microscopy of Transfected Cells
2
Olfactory Brain Structure Analysis via RNAscope
3
Olfactory Brain Structure Analysis via RNAscope
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We dissected the brain of P14–21 C57BL/6NCrl mice at ZT3.5 or ZT15.5, removed the olfactory bulbs, cerebellum and temporal lobes and fixed it with 4% PFA/PBS overnight at 4°C. The brain was then cryo-protected in 30% sucrose PBS at 4°C for 48 hour and stored in PBS for no more than a week. To prepare slices for RNAscope, we separated the two hemispheres, embedded them in agar and prepared 40 μm thick slices using a vibrating blade microtome (VT1200S, Leica Microsystems, Buffalo Grove, IL). The slices were post-fixed in 4% PFA/PBS for 30 min at room temperature (RT) and mounted onto Superfrost plus microscope slides. Mounted slices were used for fluorescence in situ hybridization (FISH) using an RNAscope multiplex fluorescent assay (Advanced Cell Diagnostics, Newark, CA) according to manufacturer instructions, using Mm-Per2-C1 and Mm-Arntl-C2 RNA probes and Opal 520 and Opal 570 dyes (Akoya Biosciences, Menlo Park, CA). DAPI Fluoromount G was used as the mounting medium (Cat# 0100–02; SouthernBiotech, Birmingham, AL). The presence of Per2 and Arntl transcripts was assessed using a confocal microscope (Zeiss LSM710) equipped with a Plan-Apochromat 63X/1.4NA oil objective. Image size was set to 1024×1024 pixels and represented the average of 8 consecutive frames.
4
Chemotactic Response of VEGFR2-Expressing Endothelial Cells
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This was performed as described44 . Briefly, ECD-VEGFR2-EYFP overexpressing ECs were seeded at 3.0 × 106 cells/mL in μ-slide chemotaxis chambers (IBIDI, Martinsried, Germany) and incubated in complete medium for 24 hours. Cells were then stimulated with gremlin11 (link) and VEGF-A (100 ng/mL)45 (link) and observed during gradient formation. After 2 hours, cells were photographed under a Zeiss Axiovert 200 M epifluorescence microscope equipped with a Plan-Apochromat 63x/1.4 NA oil objective. Z-stack images acquired using ApoTome imaging system were elaborated through AxioVision Extended Focus module (Zeiss Axiovert 200 M system).
5
Gremlin-induced VEGFR2 phosphorylation and ROS
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HUVECs or VEGFR2-overexpressing ECs were treated with gremlinWT or gremlinC141A, fixed and decorated either with anti-phospho-VEGFR2 rabbit antibody (Y1175, Cell Signaling Technology), followed by Alexa Fluor 488 anti-rabbit IgG (Invitrogen Life Technologies) or anti-phospho-Tyr mouse antibody (4G10, Upstate Biotechnology, Inc.). Cells were alternatively analysed using Axiovert 200M epifluorescence microscope equipped with Plan-Apochromat 63X/1.4 NA oil objective (Zeiss) or subjected to FACS analysis ( MACSQuant, Miltenyi Biotec) respectively. Intracellular ROS levels were assessed by measuring fluorescence intensity in HUVEC cells suspended in serum-free medium and loaded with the redox-sensitive dye DCFH-DA (5 μM, Molecular Probe, Life Technologies), as previously described [16 ]. Cells were then washed, stimulated with gremlinWT or gremlinC141A and finally subjected to FACS analysis. FACS analyses were performed with MACSQuant Analyzer (Myltenyi Biotec) and data were analysed with FlowJo software.
6
Structured Illumination Microscopy Protocol
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The ZEISS Elyra PS1 system was used with standard filter sets and laser lines with a Plan-APOCHROMAT, 63x/1.4 NA oil objective using the structured illumination microscopy (SIM) mode of the system. SIM imaging was performed using five grid rotations (0.51 mm grid) for 15 Z-planes with a 0.110 µm spacing. For SIM image reconstruction, the ZEN black software (MicroImaging, Carl Zeiss) was used with the following “method” parameters:
Nine different locations across the entire growth area of a well were imaged. Each vibration treatment was independently repeated three times, resulting in a total of 27 images per condition for analysis. All the acquired images can be found inSupplementary Raw Data S2 .
Processing: manual;
Noise filter: −4;
SR frequency weighting: 1;
Baseline cut, sectioning: 100/83/83;
PSF: theoretical;
Output: SR-SIM.
Nine different locations across the entire growth area of a well were imaged. Each vibration treatment was independently repeated three times, resulting in a total of 27 images per condition for analysis. All the acquired images can be found in
7
Immunofluorescence Analysis of Adipocyte Markers
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Cells were seeded on glass coverslips and fixed in 4.0% paraformaldehyde (PFA)/2.0% sucrose in PBS, permeabilized with 0.5% Triton-X100, and saturated with goat serum in PBS. Then, cells were incubated with UCP-1 (sc-6529, SantaCruz, CA, USA), PPARγ (sc-7273, SantaCruz), and ACRP30 (sc-26497, SantaCruz) antibodies. Cells were analyzed using a Zeiss Axiovert 200M epifluorescence microscope equipped with a Plan-Apochromat 63X/1.4 NA oil objective.
8
Visualizing VE-cadherin and Neuropilin-1 Colocalization
9
Analyzing VE-cadherin Interactions in HUVECs
10
Gremlin Regulates VEGFR2 Activation
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Coverslips were coated with recombinant gremlin at indicated doses, and GM7373-VEGFR2 cells were allowed to adhere for 1 h. To prepare ventral plasma membranes, (VPMs) cells were washed and squirted over by a jet of ice-cold water. The remaining VPMs were fixed and analyzed for VEGFR2 activation with anti-pVEGFR2 antibody by immunofluorescence [25 (link)]. VPMs were photographed using an Axiovert 200 M epifluorescence microscope equipped with a Plan-Apochromat 63X/1.4 NA oil objective (Zeiss, Oberkochen, Germany). Images were analyzed by FiJi software (http://rsbweb.nih.gov/ij , accessed on 15 November 2021).
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