Confocal images were acquired using an Olympus Fluoview FV1000XY, FV10i or FV1200 confocal microscopes and Olympus FV10-ASW v4.1 software. All imaging was performed using Olympus UPlanSApo 60X water and Olympus UPlanSApo 10X objectives. Embryos were embedded in 1% low melt agarose on cover slips for all confocal imaging. For analysis of filopodia dynamics, z-stacks of the leading edge of NC stream 3 in 26 hpf Tg(sox10:rfpmb) embryos injected with tp53MO or tp53MO plus fscn1aMO (n = 5 of each) were acquired every 2 minutes for 1 hour using the 60X water objective. For analysis of NC stream depth, z-stacks were acquired of NC stream 3 in 26 hpf Tg(sox10:rfpmb; sox10:h2a-gfp) embryos injected with tp53MO or tp53MO plus fscn1aMO. Cranial NC migration was imaged in 22, 25, 28 and 36 hpf Tg(sox10:GFP) embryos using a Zeiss Axiovert 200 inverted microscope configured with an Olympus DP72 camera. Widefield fluorescent images were acquired on an Olympus SZX16 microscope configured with an Olympus DP72 camera. Brightfield images were taken using a Nikon C-DSD115 microscope configured with an Olympus DP72 camera. Prism 6, ImageJ 1.46r, Adobe Photoshop CC 2014–2015, and Adobe Illustrator CC 2014–2015 were used to generate figures.
Dp72 camera
Manufactured by Olympus
Sourced in Japan, United States, Germany, Panama
The DP72 is a digital camera designed for microscopy applications. It features a high-resolution 12.8-megapixel sensor and supports various image capture modes. The DP72 is capable of capturing detailed images of samples under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Bx51 microscope, by Olympus (61 mentions)
Dp2 bsw software, by Olympus (15 mentions)
Bx51 fluorescence microscope, by Olympus (14 mentions)
Bx61 microscope, by Olympus (13 mentions)
Bx60 microscope, by Olympus (12 mentions)
Bx53 microscope, by Olympus (12 mentions)
Cellsens software, by Olympus (10 mentions)
Ix51 microscope, by Olympus (9 mentions)
Ix71 microscope, by Olympus (8 mentions)
Bx microscope, by Olympus (7 mentions)
Szx16 microscope, by Olympus (7 mentions)
Vectastain elite abc kit, by Vector Laboratories (6 mentions)
Szx16 stereomicroscope, by Olympus (6 mentions)
Bx41 microscope, by Olympus (6 mentions)
Cell f software, by Olympus (5 mentions)
Bx43 microscope, by Olympus (5 mentions)
Ix71 inverted microscope, by Olympus (5 mentions)
Diaminobenzidine substrate kit, by Vector Laboratories (5 mentions)
Bx63 microscope, by Olympus (5 mentions)
Bx51 light microscope, by Olympus (5 mentions)
369 protocols using dp72 camera
1
Imaging Cranial Neural Crest Dynamics
2
Multimodal Imaging of Cell Markers
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The surface morphology of HA-CTS nanofilm substrates was observed using field emission scanning electron microscopy (6700F; JEOL, Japan) at an acceleration voltage of 20 keV. QDs images with single information and traditional organic dyes images were acquired under an Olympus BX51 microscope equipped with an Olympus DP72 camera (Olympus Optical Co., Ltd., Tokyo, Japan). The signal intensities of CK and CD45 were quantified using the software package within the CRi Nuance multispectral imaging system (Cambridge Research & Instrumentation, Inc., Woburn, MA, USA). QDs images with multiple information on CK, vimentin and EpCAM/CD45 were acquired and unmixed using CRi Nuance multispectral imaging systems. After imaging acquisition, the information on these molecules were unmixed by the software package within CRi Nuance multispectral imaging system (Fig. 1 C1-C3). There were 2 major technical steps for this procedure: (1) Selection of targets with different spectra; and (2) Image unmixing and elimination of background noise.
3
Cellular Senescence Analysis by SA-β-gal Staining
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Cellular senescence was analyzed by SA-β-gal staining kit. Cells grown on 12‐well plates were treated with 200 µg/mL PM and 2.5 µM SRT1720 for 24 hours. According to the manufacture's instruction, cells were fixed with 1× fixative solution for 15 min and then stained overnight at 37°C with the β‐galactosidase staining solution at pH 6.0 for 15 hours. Cell staining was observed under a light microscope at magnification × 200, and digital images were acquired by Olympus BX51 microscope and a DP72 camera (Olympus Optical Co., Ltd., Tokyo, Japan). Areas measuring 0.26 mm × 0.26 mm (length × width) from each image were scanned. We selected type 8-bit, used the “adjust,” “threshold max 180,” and “measure” commands in ImageJ. The results of “% area” were used as the stained level of SA-β-gal, as previously described.29 –31 (link)
4
Quantifying Inflammatory Mediators
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Peritoneal lavage or in vitro supernatants were analyzed for TNFα, CXCL1/KC, and LTB4. TNFα and CXCL1 ELISA were performed with Duo Set kit, according to manufacturer’s protocol (R&D Systems, Minneapolis, MN, USA) and LTB4 with enzyme immuno assay (EIA) kit (Cayman Chemical, Ann Arbor, MI, USA). For the investigation of the presence of intracellular LTB4, we used the previously described Eicosacell protocol (31 (link)). Briefly, leukocytes were recovered from peritoneal cavities and immediately submitted to fixation and permeabilization with 0.5% 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (Sigma) in HBSS. After that, a common immunodetection protocol was performed using the following antibodies: the primary antibody anti-LTB4 (Cayman Chemical) or irrelevant IgG and the secondary antibody Alexa 488-labeled anti-rabbit IgG. Images were obtained using an Olympus BX51 fluorescence microscope and equipped with a Plan Apo ×100 objective and a DP72 camera (Olympus Optical, Japan) in conjunction with CellF Imaging Software (Olympus Life Science Europe, Germany).
5
Quantification of Ki67 and Pan-CK in Tumor Clones
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The QD-stained slides were observed under an Olympus BX51 fluorescence microscope equipped with an Olympus DP72 camera (Olympus Optical Co., Ltd., Tokyo, Japan) and CRi Nuance multi-spectral imaging system (Cambridge Research & Instrumentation, Inc., Woburn, MA, USA), at the excitation wavelength of 330–385 nm by ultraviolet light. A spectral cube for each slide was captured by CRi Nuance systems under the same conditions at low magnification (×20). The QD fluorescent signal for each cube was analyzed by CRi Nuance software package according to the manufacturer's instructions. The quantified fluorescence signals of Ki67 and pan-CK were calculated based on spectral unmixing. The ratio of Ki67 total fluorescence signal values to pan-CK total fluorescence signal values was regarded as the average expression intensity of Ki67. The quantified fluorescence signals of pan-CK were used to define the total number of cancer cells in clones. The clones were divided into three types based on the cell number in each clone, including small clones containing 14–49 cells, medium clones containing 50–100 cells, and large clones containing >100 cells.
6
Immunohistochemical Staining of FAP in CAFs
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Routine IHC method was performed for the staining of FAP. The primary antibody was rabbit anti-human monoclonal antibody against FAP (ab227703, Abcam, UK, dilution 1/200), with corresponding horseradish peroxidase (HRP) conjugated secondary antibody (ab6721, Abcam, UK, dilution 1/200). The FAP positive CAFs were indicated by both morphological features and the IHC reaction results. The reaction products were visualized with diaminobenzidine (DAB, DAKO, Denmark). Then the slides were evaluated by two senior pathologists, who were blinded to the patients’ clinical features and outcomes. A consensus was achieved using a multi-headed microscope in case of discrepancy. In brief, at least 4 standard-compliant vision fields of FAP expression (magnification, × 200) per patient was considered to be adequate, with no focus on hotspots. The digital images were captured under Olympus BX51 fluorescence microscope equipped with Olympus DP72 camera (Olympus Optical Co., Ltd., Tokyo, Japan). Identical settings were used for every photograph, so as to minimize the selection bias.
7
Imaging Insect Ommatidia Distortion
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Light microscopy images were viewed using the stereo-motorized light microscope model Olympus SZX16 (Olympus Optical) attached with an Olympus DP72 camera (Olympus Optical). Images were taken with CellSens Dimesion version 1.5 (Olympus Optical). All light microscopy images have maximum magnifications of 11x.
For scanning electron microscopy (SEM), ten progenies from each group were fixed overnight at 4°C in McDowell-Trump fixative (Sigma-Aldrich), containing 4% formaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer (Sigma) (pH 7.2). The specimens were washed in phosphate buffer three times before being post-fixed in 1% (w/v) osmium tetroxide (Sigma-Aldrich) at 25°C for an hour. The specimens were then washed with distilled water and dehydrated in a series of ethanol; 50%, 75%, 95% and 100% ethanol for 15 minutes each. The dehydrated specimens were immersed in hexamethyldisilazane (HMDS) (Sigma) for 10 minutes. The specimens were air-dried in a desiccator overnight. Dried specimens were then mounted, and gold coated to be viewed with SEM (SU8010; Hitachi Ltd., Tokyo, Japan). Degree of ommatidia distortion was obtained from each image using a computational method called Flynotyper (https://flynotyper.sourceforge.net) through Image J that calculates a phenotypic score (P-value) (Iyer et al., 2018 (link)(Iyer et al., , 2016)) (link).
For scanning electron microscopy (SEM), ten progenies from each group were fixed overnight at 4°C in McDowell-Trump fixative (Sigma-Aldrich), containing 4% formaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer (Sigma) (pH 7.2). The specimens were washed in phosphate buffer three times before being post-fixed in 1% (w/v) osmium tetroxide (Sigma-Aldrich) at 25°C for an hour. The specimens were then washed with distilled water and dehydrated in a series of ethanol; 50%, 75%, 95% and 100% ethanol for 15 minutes each. The dehydrated specimens were immersed in hexamethyldisilazane (HMDS) (Sigma) for 10 minutes. The specimens were air-dried in a desiccator overnight. Dried specimens were then mounted, and gold coated to be viewed with SEM (SU8010; Hitachi Ltd., Tokyo, Japan). Degree of ommatidia distortion was obtained from each image using a computational method called Flynotyper (https://flynotyper.sourceforge.net) through Image J that calculates a phenotypic score (P-value) (Iyer et al., 2018 (link)(Iyer et al., , 2016)) (link).
8
Chromosome Spread Analysis in MEFs
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Chromosome spread in MEFs was performed as described 35 (link), 36 (link). Briefly, the MEF cells were incubated with 100 ng/ml colcemid for 2 hours. The hypotonic treatment was carried out for 20 min at RT in 0.56% KCl. The cells were then transferred to methanol:acetic acid (3:1) for fixation. The fixation was repeated twice. All the chromosome spreads were stained with Giemsa and chromosome number and structure were examined under a 100X lens with a Leica microscope, equipped with a DP72 Olympus camera and CellSense software.
9
Cell Adhesion on Biomaterial Surfaces
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To analyze cell adhesion to the variety of different surfaces (fibronectin covered glass and fibronectin coated PCL fibers, fibroin fibers and fibroin fibers coated by fibronectin, rS1/9, rS2/12-Linker-RGDS, rS1/9+rS2/12-Linker-RGDS [1:1 mixture] recombinant spidroin meshes of various fiber diameters) the substrate specimens were placed into 24-well plate. Then isolated cardiac cells at the concentration of 1.5 ×10 cells/cm were seeded on them. After 12 hours cells were washed in PBS for 10 minutes by shaking on nutator, then fixed in 4% paraformaldehyde and stained by Alexa Fluor 488 Phaloidinin Conjugate for actin filaments visualization and DAPI for outlining of nuclei according to protocol described in [3 (link)]. Images of the stained specimens were captured with IX-71 inverted fluorescent microscope and DP72 Olympus camera. Then the cells were counted using ImageJ software and comparative analysis of the adhesion properties of different substrates was performed.
10
Interphase FISH for MYC, BCL6, and IGH/BCL2 Translocations
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Interphase FISH was performed on TMAs of 150 cases from the same cohort as previously described [17] (link). The Vysis LSI MYC dual color, break apart rearrangement probe, the Vysis LSI BCL6 dual color, break apart rearrangement probe and the Vysis LSI IGH/BCL2 dual fusion translocation probe (Abbott Molecular, Abbott Park, IL) were used. FISH signals were analyzed using a fluorescence microscope (Olympus BX51, Tokyo, Japan) equipped with a DP72 camera and DP2-BSW software (Olympus, Tokyo, Japan). Patient cases with break-apart signals in >10% of nuclei were considered positive for the presence of a translocation. The signal distribution was evaluated by two independent observers (Dong-Lan Luo and Jie Cheng). In case of discordant results between the two observers, a third investigator (Jie Xu) was involved.
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