Intracellular TAG content was measured using the “Triglycerides liquid” kit (Sentinel, Milan, Italy), as previously described [57 (link),58 (link)]. The absorbance was recorded at 546 nm using a Varian Cary50 spectrophotometer (Agilent, Milan, Italy). For measurement of extracellular TAG content, the culture media were processed according to the same method. Values were normalized to protein content, and data are expressed as percent TAG content relative to controls [59 (link)]. For intracellular lipid staining, cells grown on coverslips were rinsed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. Neutral lipids were stained by incubation with 1 μg/mL BODIPY 493/503 (Molecular Probes, Life Technologies, Monza, Italy) in PBS for 30 min [60 (link)]. After washing, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), 5 μg/mL (ProLong Gold medium with DAPI; Invitrogen, MA, USA). Mounted slides were examined at 10X magnification by Olympus IX53 light microscope (Olympus, Milano, Italy), equipped with the standard epifluorescence filter setup. Representative images were captured with a CCD UC30 camera (Olympus) and digital image acquisition software (CellSens Entry, Olympus).
Ix 53 light microscope
Manufactured by Olympus
Sourced in Japan, Italy
The IX 53 is a light microscope designed for a range of microscopy techniques. It features a modular design that allows for customization to meet specific research needs. The core function of the IX 53 is to provide high-quality, detailed imaging of specimens through the use of transmitted light illumination.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold medium with dapi, by Thermo Fisher Scientific (2 mentions)
Bodipy 493 503, by Thermo Fisher Scientific (2 mentions)
Varian cary 50 spectrophotometer, by Agilent Technologies (1 mentions)
Ccd uc30 camera, by Olympus (1 mentions)
Cellsens entry, by Olympus (1 mentions)
Isobutylmethylxanthine, by Cyagen (1 mentions)
Dexamethasone (dex), by Cyagen (1 mentions)
Insulin, by Cyagen (1 mentions)
Paraffin, by Leica camera (1 mentions)
Tgf β3, by Cyagen (1 mentions)
14 protocols using ix 53 light microscope
1
Quantifying Intracellular and Extracellular Triglycerides
2
Intracellular and Extracellular Triglyceride Measurement
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Intracellular TG content was measured in FaO cells using the “Triglycerides liquid” kit (Sentinel, Milan, Italy), as previously described [19 (link),49 (link)]. Absorbance was recorded at 546 nm; after chloroform evaporation TG content was determined in which results were recorded spectrophotometrically. For measurement of extracellular TG content, the culture media were processed according to the same method. Values were normalized to protein content and data are expressed as percent TG content relative to controls [50 (link)]. For intracellular lipid staining, cells grown on coverslips were rinsed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. Neutral lipids were stained by incubation with 1 μg/mL BODIPY 493/503 (Molecular Probes, Life technologies, Monza, Italy) in PBS for 30 min [40 (link)]. After washing, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, 5 μg/mL, (ProLong Gold medium with DAPI; Invitrogen). Mounted slides were examined at 10× magnification by Olympus IX53 light microscope (Olympus, Milano, Italy), equipped with the standard epifluorescence filter set up. Representative images were captured with a CCD UC30 camera and a digital image acquisition software (CellSens Entry).
3
Hematoxylin-Eosin Staining of Intestine and Brain
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Hematoxylin–eosin (H&E) staining was carried out according to the protocols described previously [35 (link)]. In brief, after designed treatment, the animals were sacrificed by cervical dislocation under anesthesia. The intestine and brain tissues were quickly dissected and fixed in fresh prepared 4% paraformaldehyde. The sections were subsequently dewaxed in xylene and dehydrated by ethanol, followed by staining with hematoxylin and eosin. After mounting with neutral balsam, the sections were observed under an Olympus light microscope (IX53, Tokyo, Japan).
4
Wound Healing Assay with Aaptamine
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Cells were seeded into 12-well plate at 105 cells per well 24 h before treatment. On the second day, cells were scraped with a 200 μL tip followed by incubation with serum-free RPMI medium containing 0, 8, 16 and 32 μg/mL aaptamine for 24 or 48 h. Photomicrographs were taken using Olympus light microscope IX 53 (Tokyo, Japan) of a same area before and 24 or 48 h after treatment. The remaining wound area was quantified with ImageJ software. The migration rate = (Wound width at the beginning of experiment − Wound width at the end of experiment for 24 or 48 h)/Wound width at the beginning of experiment (Zeng et al. 2019 (link)).
5
Lung Tissue Histological Analysis
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Part of the lung tissue was fixed with 4% formaldehyde, embedded with paraffin, and then stained with hematoxylin and eosin (H&E). The pathological changes of lung tissue were observed by an IX 53 light microscope (Olympus, Tokyo, Japan) [21 (link)].
6
Adipogenic Differentiation of MSCs
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MSCs from the third passage were seeded in a 24-well plate at a density of 2×104 cells/well, and cultured at 37°C and 5% CO2. When cells reached ~100% confluence, the medium was carefully aspirated from each well and adipogenic induction medium was added, which contained 10−6 M dexamethasone, 0.5 µM isobutylmethylxanthine and 10 ng/ml insulin (Cyagen Biosciences, Santa Clara, CA, USA). α-MEM supplemented with 10% FBS served as the negative control. The medium was replaced every 2–3 days. On day 14, the MSCs that committed to the adipogenic lineage were filled with lipid-rich vacuoles and were identified by staining with Oil red O. The cells were observed under an IX53 light microscope (Olympus Corporation, Tokyo, Japan).
7
Osteogenic Differentiation of MSCs
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MSCs at the third passage were seeded in a 24-well plate at a density of 3×103 cells/well and cultured at 37°C and 5% CO2. When cells were ~60% confluent, the medium was carefully aspirated and osteogenic induction medium containing 10−7 M dexamethasone, 10 mM β-glycerol phosphate and 50 µM ascorbate-2-phosphate (Cyagen Biosciences) was added. α-MEM supplemented with 10% FBS served as the negative control. The medium was replaced every 2–3 days. After 3 weeks of differentiation, the cells were fixed with 4% formalin, stained with Alizarin red and observed under an IX53 light microscope (Olympus Corporation).
8
Hoechst 33258 Staining for Apoptosis
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Apoptosis was observed using Hoechst 33258 staining. The prepared paraffin sections were deparaffinized, hydrated and then washed with PBS twice. Finally, slices were stained with Hoechst 33258 (10 µg/mL in PBS) at 37 ℃ for 15 min in the dark. Then the slices were sealed, and observed under an IX 53 light microscope (Olympus, Tokyo, Japan) [38 (link)].
9
Histological Analysis of LPS-induced Zebrafish
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Twelve hours after LPS-microinjection, the zebrafish were fixed immediately with 4% (w/v) paraformaldehyde overnight at room temperature, dehydrated in graded ethanol, embedded with paraffin (Leica, Wetzlar, Germany), and 4-µm-thick sections were cut. After being deparaffinized and stained with H&E (Yuanye Biotech, Shanghai, China), each section was observed under an IX 53 light microscope (Olympus, Tokyo, Japan).
10
BHK-21 Cells: GRE and Heparin Infection
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BHK-21 cells were grown in a 6-well plate at 37 °C overnight. After 1 h of infection with GRE or heparin at 37 °C, the cells were washed twice with PBS and cultured in RPMI-1640 supplemented with 2% FBS at 37 °C for another 4 d. The CPE was visualized under an IX 53 light microscope (Olympus, Tokyo, Japan).
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