Part of the femora was decalcified in 10% EDTA (pH 7.4) for 4 weeks and then embedded in paraffin. Four-micrometer-thick longitudinally oriented along the defect sections were used for staining. The specimens were stained with hematoxylin and eosin (H&E) according to a standard protocol, viewed under a light microscope and the stained areas were quantified using a BI-2000 medical image analysis system (Chengdu TME Technology Co, Ltd., Chengdu, China). Vascular endothelial growth factor (VEGFA) and recombinant human bone morphogenetic protein-2 (BMP-2) staining were used to quantify the expression of osteogenesis and vascularization factors in the defect area. In brief, fresh bone sections were stained with individual primary antibodies to rats VEGFA (Abcam, ab206887, 1:100) and BMP-2 (Abcam, ab214821, 1:100), overnight at 4 °C. Subsequently, the secondary antibodies conjugated with fluorescence (Jackson Immuno Research, 415-605-166, 1:500; 315-605-003, 1:250) were used at room temperature for 1 h while avoiding light and observed under a confocal microscope (FLUOVIEW FV300, Olympus). Calcein double labeling in undecalcified bone slices was observed under a fluorescence microscope (FLUOVIEW FV300, Olympus) to quantify bone mineralization in the defect area.
Fluoview fv300
Manufactured by Olympus
Sourced in Japan, United States, Italy
The Fluoview FV300 is a confocal laser scanning microscope system designed for high-resolution fluorescence imaging. It features a scanning unit, laser sources, and a control software to capture and process digital images.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (8 mentions)
Alexa 594 conjugated goat anti mouse igg, by Thermo Fisher Scientific (3 mentions)
Alexa 488 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Dcf da, by Thermo Fisher Scientific (2 mentions)
Normal goat serum (ngs), by Jackson ImmunoResearch (2 mentions)
Histoclear, by National Diagnostics (2 mentions)
Alexa 594 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Alexa 488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Cy2tm conjugated affini pure donkey anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
Cy3tm conjugated affini pure donkey anti mouseigg, by Jackson ImmunoResearch (2 mentions)
Cy5tm conjugated affini pure donkey anti goat igg, by Jackson ImmunoResearch (2 mentions)
Rabbit anti human cd31, by Santa Cruz Biotechnology (2 mentions)
Mouse anti α smooth muscle actin, by Merck Group (2 mentions)
Fvs psu ix2 ucb, by Olympus (2 mentions)
Ix71 microscope, by Olympus (2 mentions)
Fluorescent mounting medium, by Agilent Technologies (2 mentions)
Fluorescent microscope, by Olympus (1 mentions)
Ab36861, by Abcam (1 mentions)
Ab34712, by Abcam (1 mentions)
70 protocols using fluoview fv300
1
Quantifying Bone Regeneration Factors
2
Subcellular Localization of TaABF3 in Wheat
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Prof. Zhensheng Kang’s Lab (Northwest A&F University, China) provided the Triticum aestivum cv. Chinese spring, which was used in the functional analysis of TaABF3. The full-length CDS sequence of TaABF3 was amplified using PCR from the wheat cv. Chinese Spring with specific primers for the subcellular localization assay of TaABF3. This was then placed into the binary vector pCAMV35S::GFP, in between BamH I and Xba I, to find the subcellular localization of TaABF3. Sequencing was used to obtain positive clones, and the wheat mesophyll protoplasts were obtained from the constructs using the methods previously described [50 (link)]. A confocal microscope (Olympus, FluoViewTM FV300, Japan) was used to assess GFP fluorescence.
3
Subcellular Localization of TaABF3 in Wheat
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Triticum aestivum cv. Chinese spring was identi ed and obtained from the Prof. Zhensheng Kang's Lab (Northwest A&F University, China) and was used to functional analysis of TaABF3. For subcellular localization assay of TaABF3, we ampli ed the full-length CDS sequence of TaABF3 from wheat cv.
Chinese Spring by PCR using speci c primers and inserted into the binary vector pCAMV35S::GFP between Xba I and BamH I to identify the subcellular localization of TaABF3. We then identi ed positive clones by sequencing, while the construct was transformed into wheat mesophyll protoplastsas previously described [50] . The uorescence of GFP was observed using a confocal microscope (Olympus, FluoViewTM FV300, Japan).
Chinese Spring by PCR using speci c primers and inserted into the binary vector pCAMV35S::GFP between Xba I and BamH I to identify the subcellular localization of TaABF3. We then identi ed positive clones by sequencing, while the construct was transformed into wheat mesophyll protoplastsas previously described [50] . The uorescence of GFP was observed using a confocal microscope (Olympus, FluoViewTM FV300, Japan).
4
Intracellular ROS Measurement by DCF-DA
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Intracellular ROS were measured fluorometrically using 2′,7′-dichlorofluorescein diacetate (DCF-DA) (Molecular probes, Eugene, OR, USA), which permeates cells easily and hydrolyzes to DCF after interacting with intracellular ROS (Lee et al., 2016 (link)). Cultured cells were washed twice with HEPES controlled salt solution (HCSS) incubated with 10 μM DCFDA and 20% Pluronic F-127 for 30 min, and washed with HCSS, as described previously (Lee and Jung, 2012 (link)). Subsequently, cells were observed under a fluorescent microscope (Olympus, Tokyo, Japan). DCF fluorescence intensities were obtained in Fluoview FV300 software (Olympus Corporation, Shinjuku, Tokyo, Japan).
5
In Vivo Intravital Scaffold Degradation
6
Characterization of NP Phenotype in CFU-S
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The NP phenotype of CFU-S40 (link) from WT mice was confirmed double-positive for type II collagen and aggrecan by immunocytofluorescence (Supplementary Fig. S2a ). Hereafter, the NP phenotype of the cells was judged with CFU-S, in both WT and Casp-3 KO mice. The slides were fixed with 4% paraformaldehyde (PFA) for 48 h followed by 1% PFA for 20 min at room temperature. The samples were washed with phosphate buffered saline (PBS) for 5 min, three times, and permeabilized with PBS containing 0.1% Triton X-100 for 30 min. Next, the slides were blocked with 1% bovine serum albumin containing PBS for 60 min at room temperature, then incubated with primary antibodies against type II collagen (1:400; ab34712; Abcam) and aggrecan (1:100; ab36861; Abcam) at 4 °C. They were rinsed with PBS for 5 min, three times, and incubated with anti-rabbit IgG conjugated with Alexa 488 (1:200; A-21206; Thermo Fisher Scientific) and anti-rabbit PE (1:200; 711-116-152; Jackson ImmunoResearch Laboratories, Baltimore Pike, PA, USA) for 60 min. After the samples were washed with PBS and distilled water, the nuclei were counterstained with DAPI. The CFU-S were imaged using confocal laser microscopy (Olympus Fluoview FV300, Tokyo, Japan).
7
Immunofluorescence Staining of Bone Tissue
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Immunofluorescence staining was conducted as described in previous studies (Rached et al., 2010 (link); Yang et al., 2017 (link)). Fresh bone tissues were collected and fixed immediately in 4% paraformaldehyde solution at 4°C for 4 h. Subsequently, the bone tissues were placed in 0.5 M ethylene diamine tetraacetic acid solution (pH 7.4) for decalcification at 4°C for 48 h, followed by dehydration with 20% sucrose and 2% polyvinylpyrrolidone (PVP) solution for 1 day until they descended to the bottom. Next, 8% gelatin (porcine; Sigma, G2500), 20% sucrose, and 2% PVP (Sigma, PVP360) were mixed and dissolved with PBS at 60°C. Finally, the tissues were embedded as described above. Forty-micrometer-thick, longitudinally oriented bone sections were sliced and stained with primary antibodies of mouse CD31 (Abcam, ab28364, 1:100) and endomucin (Santa Cruz, V.7C7, 1:50) overnight at 4°C. Next, the sections were incubated with secondary antibodies (Jackson ImmunoResearch, 415-605-166, 1:500; 315-545-003, 1:500) at 37°C for 1 h, away from light. Polyclonal goat IgG (R&D Systems, AB-108-C) and monoclonal rat IgG2A (R&D Systems, 54447) were used as negative controls. The specimens were observed using a confocal microscope (FLUOVIEW FV300, Olympus).
8
Measurement of Intracellular ROS Levels
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We followed a method reported previously to determine ROS levels [15 (link)]. In brief, cells grown on a glass-bottomed dish were loaded with 10 μM 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate dicarboxymethyl ester (DCF-DA) in HCSS buffer containing 120 NaCl, 5 KCl, 1.6 MgCl2, 2.3 CaCl2, 15 glucose, 20 Hepes, and 10 NaOH (mM, pH 7.4) for 20 min at 37°C. DCF-DA was purchased from Molecular Probes (Eugene, OR, USA). The experiments were performed at room temperature on A confocal microscope stage and digitized using FLUOVIEW FV300 (Olympus, Tokyo).
9
Multicolor Fluorescence Microscopy for Sialidase Activity
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Images were acquired on an Olympus Fluoview FV300 laser-scanning microscope system equipped with an argon laser (488 nm), a HeNe laser (543 nm), and a diode laser (405 nm) for excitation. To visualize X-NANA (sialidase activity staining), 430- to 460-nm emissions were collected after excitation at 405 nm. FITC (505- to 525-nm emissions) and rhodamine (565-nm emissions) signals were visualized in sequential mode by 488 nm and 543 nm excitation, respectively, and standard emission filters.
10
WISH for PpTRPA1 in Bipinnaria
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Whole-mount in situ hybridization (WISH) was carried out as previously described with the modification that 4-day bipinnaria were treated with proteinase K for 2 h for experiments using the PpTRPA1 probe64 (link). To detect fluorescent signals, an HNPP Fluorescent Detection Set (Roche) was used. Coronal plane stack images were obtained by using the confocal leaser scanning microscope system (Fluoview, FV300, Olympus, Tokyo, Japan) and z-stack images were generated using Image J software. The probes for PpTRPA1 and PpTRPA basal included from 1 bp to 1812 bp and from 2663 bp to 4170 bp of the open reading frame sequences, respectively.
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