The zebrafish wild AB strain was purchased from the Zebrafish International Resource Center (Eugene, OR, USA) and maintained in our facility according to standard operational guidelines. Twenty-four hours post-fertilization (hpf) embryos were dechorionated using 2 mg/mL Pronase solution (Sigma-Aldrich, St Louis, CA, USA), following the methodology used in previous studies [36 ]. Following this, 12 embryos were transferred in 4 mL of 0.3× Danieau’s solution (fish medium) to six-well plates with or without lecithin-ceramides, and incubated for 96 h at 28 °C. At 72 hpf, images of the larvae were captured using a BZ-X710 microscope (Keyence, Tokyo, Japan). To evaluate the toxicity of the tested compounds, the larvae were exposed to the emulsions for five days. The fish medium was refreshed every other day during the experiment. The number of survivals was recorded daily. For the PC70-NR experiment, we added 1% PC70-NR or 1 ng/mL NR solution to 72 hpf zebrafish larvae and incubated these for 2 and 20 h, respectively, at 28 °C. The fish were then washed with breeding water for 18 h. Fluorescent images of the larvae were captured using a BZ-X710 microscope (Keyence).
Bz x710 microscope
Manufactured by Keyence
Sourced in Japan, United States, Germany
The BZ-X710 is a digital microscope designed for detailed observation and analysis of samples. It offers high-resolution imaging capabilities and a range of advanced features for scientific and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Bz x analyzer software, by Keyence (18 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (18 mentions)
Bz x viewer, by Keyence (10 mentions)
Bz x analyzer, by Keyence (10 mentions)
Vw 9000 video editing analysis software, by Keyence (10 mentions)
Oct compound, by Sakura Finetek (6 mentions)
Phalloidin, by Thermo Fisher Scientific (5 mentions)
T20 automated cell counter, by Bio-Rad (4 mentions)
Diff stain kit, by Electron Microscopy Sciences (4 mentions)
Collagen 1, by Corning (4 mentions)
Matlab, by MathWorks (4 mentions)
Glass bottomed dishes, by MatTek (4 mentions)
Bz x710, by Keyence (4 mentions)
Mitotracker green, by Thermo Fisher Scientific (4 mentions)
Paraformaldehyde (pfa), by Fujifilm (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (4 mentions)
Lsm 510 meta confocal microscope, by Zeiss (4 mentions)
Euthasol, by Virbac (3 mentions)
Tryple express solution, by Thermo Fisher Scientific (3 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (3 mentions)
397 protocols using bz x710 microscope
1
Zebrafish Embryo Toxicity Assay
2
Immunolabeling of Exogenous Lens Proteins
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The dissected embryo lenses were fixed in 2% PFA and incubated overnight at 4 °C, dehydrated with sucrose, and embedded in Tissue-Tek compound. Sagittal sections (12 mm) were collected and stored at −20 °C. For lenses injected with hAQP0WT or hAQPR33C, whole lens images were captured with a Keyence BZ-X710 microscope before fixation. For immunolabeling of exogenous proteins, lens sections were first incubated in phosphate-buffered saline (PBS) for 5 min, then in blocking solution (2% normal goat serum, 2% fish skin gelatin, 0.25% Triton X-100 and 1% bovine serum albumin (BSA) in PBS) for 1 h, and finally in blocking solution containing monoclonal anti-FLAG antibody (1:1000 dilution) overnight at 4 °C. Sections were washed three times, 5 min each time in PBS, and then incubated with fluorescein-conjugated donkey anti-mouse IgG against anti-FLAG (1:500 dilutions in blocking solution) for 2 h at room temperature. After three washes in PBS for 5 min each, a drop of mounting medium was added before being covered by a glass coverslip. The images of specimens were taken using a Keyence BZ-X710 microscope. Imaging acquisition conditions were kept constant for all samples.
3
3D Cell Invasion Assay with Collagen IV
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Spheroid invasion assays were performed as described previously (32 (link)). 1,000 231-GFP or 2,000 PyMT-GFP cells were seeded in round-bottom, low-attachment 96-well plates and centrifuged at 3,000 g for 3 minutes to promote spheroid formation. Spheroids were allowed to grow for three days before addition of an ECM mixture of 1 mg/mL collagen I (354236; Corning), 10 mmol/L NaOH, 7.5% 10X DMEM, and 50% DMEM with or without 20 µg/mL native human collagen IV (ab7536; Abcam). Once ECM had solidified, 50 μL of culture media was added to maintain moisture and humidity in the well. In inhibitor studies, this media included DMSO as a vehicle control or small molecule inhibitors. Spheroids were imaged in 3D on the day of ECM addition and after 4 days of growth using a Keyence BZ-X710 microscope (Keyence).
For 3D single-cell invasion assays, 20,000 231-GFP or PyMT-GFP cells were suspended in ECM mixture described above with or without 20 µg/mL collagen IV and seeded into 48-well tissue culture plates. Once ECM had solidified, 50 μL of culture media to maintain moisture and humidity in the well. In inhibitor studies, cells were treated with DMSO as a vehicle control or small molecule inhibitors 1 hour before imaging. Cells with imaged live in 3D over 16 hours using a Keyence BZ-X710 microscope and invasive speed was quantified as in reseeding experiments.
For 3D single-cell invasion assays, 20,000 231-GFP or PyMT-GFP cells were suspended in ECM mixture described above with or without 20 µg/mL collagen IV and seeded into 48-well tissue culture plates. Once ECM had solidified, 50 μL of culture media to maintain moisture and humidity in the well. In inhibitor studies, cells were treated with DMSO as a vehicle control or small molecule inhibitors 1 hour before imaging. Cells with imaged live in 3D over 16 hours using a Keyence BZ-X710 microscope and invasive speed was quantified as in reseeding experiments.
4
Quantifying Aortic Lipid Deposition
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The aortas were dissected and the adherent (adventitial) fat was gently removed. Whole aortas were opened longitudinally from the aortic arch to the iliac bifurcation, mounted en face, and stained for lipids with oil red O. Hearts were embedded in optimum cutting temperature compound (OCT) (Tissue-Tek, Sakura, Torrance, California) and serial 7-μm-thick cryosections from the aortic sinus were mounted and stained with oil red O and hematoxylin. Six frozen aortic root cross sections for oil red O stain or hematoxylin were captured with BZ-X710 microscope (Keyence, Itasca, Illinois) digital camera. Image analysis was performed by a trained observer blinded to the genotype of the mice. Lesion areas were quantified with image analysis software using a BZ-X710 microscope (Keyence). The lesion area in the aorta en face preparations was expressed as a percent of the aortic surface area, as previously reported (44 (link)). Necrotic core was measured by hematoxylin-eosin staining and quantified with image analysis software.
5
Muscle Fiber Cross-Sectional Area Analysis
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Dissected soleus muscle was soaked in optimal cutting temperature compound (Sakura Finetek, Tokyo, Japan), and then immediately frozen in isopentane iced with liquid nitrogen and stored at −30 °C until sectioning. Frozen muscle samples were sectioned at a thickness of 12 μm, air dried, and stored at −30 °C. Images were captured with the BZ-X710 microscope (Keyence, Osaka, Japan).
To verify the CSA of muscle fibers, the muscle sections were incubated overnight with Laminin alpha2 antibody to stain the sarcolemma.
Subsequently, the sections were incubated with Alexa for 90 min and then washed with Tris-buffered saline with Tween 20 (TBS-T, 50 mM Tris (pH = 7.4), 138 mM NaCl, 2.7 mM KCl, and 0.05% Tween 20) three times for 5 min. The stained sections were observed under the BZ-X710 microscope and CSA analysis was carried out using the BZ-X Analyzer (Keyence).
To verify the CSA of muscle fibers, the muscle sections were incubated overnight with Laminin alpha2 antibody to stain the sarcolemma.
Subsequently, the sections were incubated with Alexa for 90 min and then washed with Tris-buffered saline with Tween 20 (TBS-T, 50 mM Tris (pH = 7.4), 138 mM NaCl, 2.7 mM KCl, and 0.05% Tween 20) three times for 5 min. The stained sections were observed under the BZ-X710 microscope and CSA analysis was carried out using the BZ-X Analyzer (Keyence).
6
Immunofluorescent Staining of Phospho-SMAD3
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Tumor samples were sectioned with 7 μm thickness and fixed in 4% paraformaldehyde for 10 min. The tumor sections were washed and then blocked with Blocking One reagent (#03953‐95; Nacalai Tesque). The blocked specimens were incubated with primary antibodies in Blocking One reagent at 4 °C overnight. After washing the plates, the samples were incubated with secondary antibody in Blocking One reagent at 37 °C for 30 min. Washed plates were mounted with 4′,6‐diamidino‐2‐phenylindole (DAPI) Fluoromount‐G (#0100‐20; SouthernBiotech, Birmingham, AL, USA) and analyzed using a BZ‐X710 microscope (Keyence). Nonspecific signals with strong intensity were removed. The following antibodies were used: SMAD3 phospho‐S423/phospho‐S425 antibody (#600‐401‐919; Rockland Immunochemicals, Limerick, PA, USA), rabbit IgG‐UNLB (isotype control, #0111‐01; SouthernBiotech), and goat anti‐rabbit IgG (H + L) cross‐adsorbed secondary antibody, Alexa Fluor 488 (#A‐11008; Invitrogen, Thermo Fisher Scientific).
7
Microscopic Evaluation of polyI:C Transfection
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To evaluate the cell state after polyI:C transfection and capture images, an IX70 microscope with DP72 microscope digital camera (Olympus, Tokyo, Japan) or a BZ‐X710 microscope (Keyence, Osaka, Japan) was used.
8
Acute Brain Tissue Preparation and Analysis
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Animals in the acute studies were sacrificed at P14. Following euthanasia, cardiac perfusion was performed with cold PBS followed by 4% paraformaldehyde mixed in PBS. Brains were extracted and placed in 4% paraformaldehyde solution overnight at 4 °C. The brains were then moved to a new vial with 30% sucrose mixed with 4% paraformaldehyde in PBS. For tissue cutting, the brains were embedded in Tissue-Plus Optimal Cutting Temperature (OCT) compound (23-730-571, Fisher Healthcare) and frozen. Brains were cut in 40 µm coronal sections using a freeze-mount cryostat. Sections from the complete brain were collected in 12-well plates kept in PBS-filled wells until histologic analysis. For Nissl staining, serial brain sections 200 µm apart were mounted on a slide and stained using cresyl violet as previously described [27 (link)]. For ventricular and lesion volume measurements, 8 serial Nissl-stained brain sections 200 µm apart and 40 µm thick were used to reconstruct the total lesion volume. 4 × magnification images of each slice were acquired using a Keyence BZ-X710 microscope (Keyence Co., Itasca, IL, USA). Lesion and ventricular areas were calculated using NIH ImageJ (FIJI) in a blinded fashion.
9
Fluorescence Microscopy of Actin and Cortactin
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Cells were imaged on a Keyence BZ-X710 microscope (Keyence, Itasca, IL) using the relevant filter cubes for DAPI (blue filter), Actin (red filter), Cortactin (green filter). All images were acquired with the same magnification (100X, oil immersion), exposure time, and illumination intensity. Images were quantified and processed using ImageJ software.
10
Electrolytic Lesion and DREADD Verification
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At the conclusion of the experiment, rats were anesthetized and administered electrolytic lesions at the recording sites via direct current stimulation (20 μA for 20 seconds), Fig. 3A .Rats were euthanized 3 days later by sodium pentobarbital (Euthasol,0.8 mL, i.p.; Virbac, Fort Worth, TX) and brains were extracted via transcardial perfusion with phosphate buffered saline (PBS) followed by 10% buffered formalin acetate and post-fixed in this solution for 24 hours followed by 30% sucrose cryoprotection. Tissue was prepared in 40-μM thick coronal sections and either Nissl stained for verification of electrode placement (Fig. 3B ) or cover slipped with DAPI mounting medium (Prolong gold, Invitrogen, Carlsbad, CA) and amplified with NMDAR1 Polyclonal Antibodies (Invitrogen for Thermo Fischer Scientific) for DREADDs verification, visualized using a BZ-X710 microscope (Keyence, Itasca, IL), and analyzed with BZ-X Viewer software. DREADDs expression (visualized by magenta fluorescence) was determined by matching histological sections to a standard rat brain atlas [66 ], Fig. 3C .
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