ORO staining was conducted to identify the adipogenic committed cells. Briefly, the BMSCs were fixed using 4% PFA solution, dyed with ORO solution (Cyagen Biosciences, China) for 30 min, and then observed through an inverted light microscopy (Nikon, Japan). Finally, the representative images were acquired and the number of adipocytes was obtained.
Inverted light microscopy
Manufactured by Nikon
Sourced in Japan
Inverted light microscopy is a type of optical microscope that illuminates the specimen from below, allowing the observation of samples that are not suitable for traditional upright microscopy. It provides a clear and detailed view of the sample, enabling detailed analysis and observation.
Automatically generated - may contain errors
Lab products found in correlation
Oro solution, by Cyagen (1 mentions)
Flow cytometry, by BD (1 mentions)
Fitc labeled annexin 5 pi apoptosis detection kit, by Keygen Biotech (1 mentions)
Xcam α, by Olympus (1 mentions)
Digital camera, by Nikon (1 mentions)
Nis elements software, by Nikon (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Tryptose phosphate broth, by Merck Group (1 mentions)
Yeast extract, by Acumedia (1 mentions)
7 protocols using inverted light microscopy
1
Adipogenic Commitment Evaluation via ORO
2
Annexin V Apoptosis Assay of Compounds
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Cell morphological changes were observed with inverted light microscopy (NIKON, Tokyo, Japan). To identification of the apoptotic induction effect of compounds 6 and 14 , a FITC-labeled Annexin V/PI apoptosis detection Kit (Keygen, Nanjing, China) was used according to the manufacturer’s instructions. Briefly, HL60 cells were exposed to vehicle control (DMSO, <0.1%), compounds 6 (12 μM) and 14 (12 μM). After 48 h, cells were harvested and washed with PBS and resuspended in binding buffer, and then, AnnexinV-FITC and PI were added. After staining for 15 minutes, the cells were immediately analyzed using flow cytometry (Becton Dickinson, USA).
3
Passage-1 Cell Morphology Analysis
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Phase contrast microscopy (PCM) was performed using inverted light microscopy (Nikon, Japan) to determine passage-1 (P1) morphology of cells cultured on the T75 plastic flasks. The cells were observed at X10/0.25 objective lens (Olympus, USA) and images captured using Xcam-α digital camera (Olympus) up to three image fields/experimental group.
4
Histomorphometric Analysis of Tendon-Bone Healing
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After the micro-CT scan, the FTGTC samples were fixed in 4% neutral buffer formalin for 48 h and then decalcified with 10% EDTA for approximately 4 weeks. The samples were embedded with paraffin, and 5 μm-thick paraffin sections were obtained in the direction perpendicular to the longitudinal axis of the bone tunnel. The sections were stained with hematoxylin-eosin (H&E) and safranin O-Fast green (S&F) staining, and were visualized with inverted light microscopy (Nikon Co., Japan). Histomorphometric analysis was performed to assess the tendon-bone tunnel interface, the width of the tendon-bone interface, the percentage of graft-bone contact, and the healing pattern within the interface of the tendon-bone tunnel. Furthermore, immunohistochemical staining was performed to evaluate collagen formation and detect osteocalcin deposition. Sections were incubated with the associated primary antibodies overnight at 4 °C and subsequently incubated with the secondary antibodies for 1 h at room temperature. Then, the stained sections were developed in diaminobenzidine solution and counterstained with hematoxylin. Slides were observed, and digital images were captured using a Nikon light microscope equipped with a Nikon digital camera and NIS-Elements software (Nikon, Japan).
5
PEDV Virus Neutralization Assay
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Plasma samples of piglets were heated at 56 °C for 30 min to inactivate complement prior to use. For each well, mixtures containing 50 μL of PEDVPT-P5 virus (50 viral particles) and 50 μL of 2-fold diluted plasma samples in PI medium were incubated at 37 °C for 1 h before applying to Vero cells (2 × 104/well). After incubation with the virus-plasma mixture for 1 h, Vero cells were washed twice and maintained in PI medium for 24 h. Cytopathic effects were detected using inverted light microscopy (Nikon, Tokyo, Japan). The neutralizing titer was defined as the highest dilution without CPE.
6
Cell Morphology Assessment of Zephycandidine A
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The cell morphological assessment was performed after HL-60 cells were treated with 0, 3 and 6 μM of zephycandidine A (1 ) for 48 hours. Cell morphological changes were observed with inverted light microscopy (NIKON, Tokyo, Japan).
7
Neutralizing Antibody Assay for PEDV
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The plasma samples of piglets were first incubated at 56 °C for 30 min to inactivate the complement. The neutralizing assay was performed in accordance with the procedure which was previously published [9 (link),35 (link)]. The plasma samples were diluted from 10-fold to 320-fold by freshly prepared post-inoculation (PI) medium containing DMEM (Gibco, Gaithersburg, MD, USA) supplemented with 0.3% tryptose phosphate broth (Sigma, St. Louis, MO, USA), 0.02% yeast extract (Acumedia, Lansing, CA, USA), and 10 µg/mL of trypsin (Gibco). The mixture of PEDVPT-P6 and 7 (200 TCID50/mL) and diluted plasma samples at equal volume were added to each well and incubated at 37 °C for 1 h and applied to Vero cell-coated microplates for another hour of incubation. Then, these microplates were washed and maintained in PI medium for 24 h. The cytopathic effect (CPE) was identified under an inverted light microscopy (Nikon). The neutralizing titers of the plasma samples were determined as the highest dilution without CPE.
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