Images were acquired with a Zeiss LSM 710 inverted confocal laser scanning microscope and a Keyence BZ-X710 fluorescence microscope. To count the number of ACR2-expressing cells, a 10× objective lens was used in the Zeiss LSM 710 with 405-, 488-, and 561-nm argon lasers. To verify positions, a 4× objective lens was used in the Keyence BZ-X710 with DAPI, GFP, and Cy5 filter cubes (Keyence). The ImageJ software47 (link) was used for adjustment of brightness and contrast, and quantification of ACR2-expressing cells (Cell Counter plugin). Three brain slices on the right side of 3 different mice were used for cell counting. Cells expressing TH or ACR2, as well as cells expressing both TH and ACR2 simultaneously, were counted.
Bz x710
Manufactured by Keyence
Sourced in Japan, United States, United Kingdom, Germany
The BZ-X710 is a digital microscope that captures high-resolution images and video. It features a 12MP CMOS image sensor and can magnify specimens up to 4,000x. The microscope is equipped with LED illumination and supports a range of objective lenses.
Automatically generated - may contain errors
Lab products found in correlation
Bz x analyzer, by Keyence (29 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (17 mentions)
Hoechst 33342, by Thermo Fisher Scientific (17 mentions)
Lsm 880, by Zeiss (12 mentions)
Bz x viewer, by Keyence (10 mentions)
Lsm 710, by Zeiss (8 mentions)
Dapi fluoromount g, by Southern Biotech (7 mentions)
Oct compound, by Sakura Finetek (7 mentions)
Bz x analyzer software, by Keyence (7 mentions)
Blocking one, by Nacalai Tesque (6 mentions)
Ezview plate, by AGC Techno Glass (6 mentions)
Paraformaldehyde (pfa), by Fujifilm (6 mentions)
Fv1000, by Olympus (6 mentions)
Lsm 780, by Zeiss (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (6 mentions)
Lsm 700, by Zeiss (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions)
Op 87763, by Keyence (5 mentions)
Bz analyzer, by Keyence (5 mentions)
Fluoview fv10i, by Olympus (5 mentions)
728 protocols using bz x710
1
Quantification of ACR2-Expressing Cells in Mouse Brain
2
Osteoclast Formation and Activation on ZOL/β-TCP
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To examine the effects of the ZOL/β-TCP for osteoclast formation and activation, mBMSCs were cultured on each ZOL/β-TCP disc in accordance with the schedule of Figure 6 . In brief, isolated mBMSCs (5.0 × 105 cells/well) were cultured on ZOL/β-TCP discs in differentiation inducer medium, α-MEM with 10% FBS, 0.1% antibiotics, 30 ng/mL M-CSF and 50 ng/mL receptor for nuclear factor kappa B ligand (RANKL, Peprotech Inc., Cranbury, NJ, USA), for 5, 10 days at 37 °C. In this examination, we used the ZOL/β-TCP disc, immersing in culture medium for 24 h before seeding mBMSCs. The medium was exchanged every 2, 3 days. After culturing, to confirm osteoclast formation, cells were stained with Alexa Fluor® 488-labeled phalloidin (Invitrogen, Carlsbad, CA, USA) for F-actin and DAPI (Dojindo, Kamimashiki, Kumamoto, Japan) for nuclei. Cells were washed with PBS (pH 7.3) and examined by observation with fluorescence microscopy (BZ-X710, Keyence, Osaka, Osaka, Japan). To confirm osteoclast activation, cells were performed with tartrate-resident acid phosphate (TRAP) staining using TRAP/ALP Stain Kit (Wako Pure Chemical, Osaka, Japan) and observed by phase microscopy (BZ-X710, Keyence, Osaka, Osaka, Japan). We analyzed three indexes from the results of observation: the osteoclasts area, cells size, and TRAP-positive area by BZ-X Analyzer (BZ-H3A (ver. 1.31), Keyence, Osaka, Osaka, Japan).
3
Dystrophin Expression in DYS-HAC Pigs
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After DYS-HAC-cloned pigs were euthanized under general anesthesia, the skeletal muscles (biceps femoris) were dissected, mounted on cork bases using tragacanth gum (Fujifilm Wako Pure Chemical, Osaka, Japan), and frozen by isopentane cooled in liquid nitrogen. Histological analyses were performed on 8-μm-thick frozen sections. After the observation of EGFP expression in the frozen section under fluorescence microscopy (BZ-X710, Keyence, Osaka, Japan), immunofluorescence staining for dystrophin was performed using the same frozen section. The histological serial sections were stained with H&E using a standard technique. For immunofluorescence, the sections were incubated with Protein Block (X0909, Dako, Glostrup, Denmark) for 30 min at 25°C and then treated with Mouse Monoclonal Antibody Dystrophin (NCL-DYS2, 1:50 dilution, Leica Biosystems, Wetzlar, Germany), which reacts with both human and pig dystrophin, for 1 h at room temperature. After the removal of excess antibody, the sections were incubated with Alexa Fluor 594 (ab150112,1:450 dilution, Abcam, Cambridge, UK) for 1 h at room temperature. The slides were visualized under fluorescence microscopy (BZ-X710, Keyence).
4
Superoxide Production in Aortic Rings and PVAT
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The superoxide production of aortic rings and PVAT were assessed with dihydroethidium (DHE) (D11347, Invitrogen, MA, USA) as previously described [20 (link)]. The frozen sections of aortic rings both without and with PVAT were cut into 10 µm thick sections and placed on glass slides. The samples were incubated at room temperature for 30 min with DHE (2 × 10−6 mol/L) and protected from light. Images of the samples were observed using a microscopic system (BZ-X710, Keyence, Osaka, Japan) with an excitation wavelength of 540 nm and an emission wavelength of 605 nm. The fluorescence intensity of the DHE staining was measured using a BZX analyzer (Keyence, Osaka, Japan). In addition, we employed the tempol (176141, Sigma-Aldrich, MO, USA), a superoxide scavenger, in order to test the contribution of superoxide production for the aorta and PVAT. We incubated the aortic rings both without and with PVAT, with tempol (10−4 mol/L) for 1 h, and then made the slides of these samples with tempol. The samples were incubated at room temperature for 30 min with DHE (2 × 10−6 mol/L) and protected from light. Images of the samples were observed using a microscopic system (BZ-X710, Keyence, Osaka, Japan) with an excitation wavelength of 540 nm and an emission wavelength of 605 nm. The fluorescence intensity of the DHE staining was measured using a BZX analyzer (Keyence, Osaka, Japan).
5
Neonatal Rat Cardiac Myocyte Hypoxia Assay
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Neonatal rat cardiac myocytes (NRCMs) were prepared from the ventricles of 2-day-old SD rats51 (link). All experiments using NRCMs were approved by the ethics committees at the National Institutes of Natural Sciences (Japan) and performed according to approved protocol code: 23A015. NRCMs were seeded on a matrigel-coated glass bottom dish and cultured in DMEM (low glucose) supplemented with 2% FBS. Two days after plating, NRCMs were incubated under normoxia or hypoxia (1% O2) for 6 h. TT or TTS (10 µM) was added during the last 30 min of normoxia or hypoxia. Untreated cells served as controls.
For H2S2 imaging, NRCMs were incubated with 5 µM SSP4 in HBSS containing 0.04% Pluronic F-127 and 2 μg/mL Hoechst for 30 min37 (link). NRCMs were washed with HBSS three times and imaged by the BZ-X710 (Keyence). The SSP4 fluorescence intensities from the cell images were calculated using Fiji and ImageJ (National Institutes of Health). Data are expressed as mean ± standard error of the mean (SEM). Statistical evaluations were performed on GraphPad Prism with ordinary one-way ANOVA.
For H2S imaging, cells (without TT/TTS treatment) were incubated with 2.5 µM SF7-AM in HBSS containing 2 μg/mL Hoechst for 30 min. NRCMs were washed with HBSS three times and imaged by the Keyence BZ-X710. The results are shown in the Supplementary Fig.8 .
For H2S2 imaging, NRCMs were incubated with 5 µM SSP4 in HBSS containing 0.04% Pluronic F-127 and 2 μg/mL Hoechst for 30 min37 (link). NRCMs were washed with HBSS three times and imaged by the BZ-X710 (Keyence). The SSP4 fluorescence intensities from the cell images were calculated using Fiji and ImageJ (National Institutes of Health). Data are expressed as mean ± standard error of the mean (SEM). Statistical evaluations were performed on GraphPad Prism with ordinary one-way ANOVA.
For H2S imaging, cells (without TT/TTS treatment) were incubated with 2.5 µM SF7-AM in HBSS containing 2 μg/mL Hoechst for 30 min. NRCMs were washed with HBSS three times and imaged by the Keyence BZ-X710. The results are shown in the Supplementary Fig.
6
Tracing Subcortical Visual Pathways
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After imaging, each mouse was transcardially perfused with 4% paraformaldehyde, and the brain was removed and submerged in the fixative solution overnight. Thereafter, coronal sections were obtained (thickness of either 50 or 100 μm), which were subsequently examined localization of the infection site in the LPN or LGN and the distribution of LPN or LGN axons in V1. For assigning the V1 layers, the sections were stained using NeuroTrace Red (cat# N-21482, Molecular Probes). All fluorescent photographs were obtained using an epifluorescence microscope (BZX-710, Keyence). The photographs of the retrogradely labeled areas were taken by either the optical sectioning method with structured illumination (BZX-710, Keyence) or confocal microscope (A1R HD25, Nikon).
7
Quantifying Neuroglial Markers in Mouse Brain Tissue
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Mice at 16W were deeply anesthetized with sodium pentobarbital (60 mg/kg body weight, intraperitoneal injection; Somnopentyl; BCM International, Hillsborough, NJ). Brain tissue sections (40 µm) were prepared with a sliding microtome and stained using the following primary antibodies: Iba1 (1:2000, 019-19741; Fuji lm-Wako Pure Chemical, Tokyo, Japan), glial brillary acidic protein (GFAP; 1:1000, Rb-Af800; Frontier Institute, Hokkaido, Japan), and doublecortin (DCX; 1:2000, AB2253; Millipore, Burlington, MA, USA). Fluorescence intensities of Iba1 + and GFAP + cells (Fig. 2B and2E) were quanti ed with a BZX-710 microscope and its integrated software (BZX-710; Keyence, Osaka, Japan). Densities of Iba1 + , GFAP + , and DCX + cells (Figs. 2C, 2F, and2H) were counted using Photoshop CS5 (Adobe, San Jose, CA, USA).
8
Quantitative Imaging of Oxidized LDL Uptake
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The U937 cells were treated with 10 μg/mL Dil-ox-LDL in RPMI 1640 medium including 10% FCS at 37 °C in 5% CO2 for 18 h [25 (link),32 (link),36 (link)]. After twice washing with PBS gently, immunofluorescence was observed using Keyence BZ-X710 microscope and analyzed with the Keyence BZ-X710 software (Osaka, Japan). The quantification of fluorescent intensity of red color per cells was calculated as described previously [25 (link),32 (link),36 (link)].
9
Matrigel-based Tube Formation Assay with Macrophage Coculture
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35-mm dish was filled with 800 µL of Matrigel and allowed to solidify at 37°C for 30 min. Subsequently, PKH67 (green)-labeled b.End5 cells (6.0 × 105 cells) were gently seeded on top of the gel, followed by cocultured with PKH26 (red)-labeled Mφ (–), Mφ (IL-10), Mφ (IL-18), or Mφ (IL-10 + IL-18) (1.5 × 105 cells) on Matrigel. After that, 35-mm dish was placed on the stage of an All-in-One fluorescence microscope BZ-X710 (Keyence) equipped with an environmental chamber, which provided 37°C, humidity and 5% CO2 conditions. Time-lapse live-cell imaging of Matrigel tube formation assay was obtained at 1–3 µm intervals for the z-axis every 10 min over 16 h with a set of green and red emissions at original magnification 200×. Obtained images were processed and analyzed with BZ-X710 software (Keyence).
10
Autophagy Analysis of Skeletal Muscle
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For autophagy analysis of thigh skeletal muscle, C15orf48–/–/GFP-LC3 mice and GFP-LC3 control mice were maintained without food for 48 h, but with free access to drinking water. Before analysis, mice were anaesthetized and perfused through the left ventricle with 4% paraformaldehyde in PBS. Thymi were collected from normally fed mice and fixed with 4% paraformaldehyde in PBS for 4 h, followed by treatment with 15% sucrose in PBS 4 h at room temperature, and 30% sucrose in PBS overnight at 4 °C. Thymic samples were embedded in optimal cutting temperature (OCT) compound and stored at −80 °C. Sections (5-μm thickness) of the thymus in OCT compound were mounted on glass slides coated with aminosilane and fixed with ice-cold acetone for 5 min. After washing, sections were blocked with 10% goat serum and then stained with Keratin 5 or Keratin 8 together with GFP antibody (Abcam) in 10% goat serum for overnight at 4 °C. After washing and further incubation with fluorescence-labeled secondary antibodies for 1 h, sections were covered with glass coverslips. Images were acquired using a BZ-X710 microscope (Keyence). Areas of keratin 5- or 8-positive regions and muscle fibers and the number of LC3 puncta were calculated using BZ-X710 software (Keyence).
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