Nuclear morphology was detected by staining DNA using Hoechst 33342. Cells grown on collagen coated glass coverslips were fixed in ice cold methanol/acetic acid and then stained with Hoechst 33342 (5 μg/ml). After washing with deionized water, cells were mounted in 50% glycerol containing 20 mM citric acid and 50 mM disodium orthophosphate and observed under a fluorescent microscope (U-LH 100–3, Olympus).
U lh100 3
Manufactured by Olympus
Sourced in Japan
The U-LH100-3 is a laboratory equipment product from Olympus. It is a light source that provides illumination for microscopy applications. The product specifications and core function are not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Deadend fluorometric tunel system, by Promega (2 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Anti rabbit secondary antibody, by Abcam (1 mentions)
α smooth muscle actin α sma, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Ab2961, by Abcam (1 mentions)
α sma, by Abcam (1 mentions)
Pa1 822a, by Thermo Fisher Scientific (1 mentions)
Tecnai f20, by Thermo Fisher Scientific (1 mentions)
Xe7 atomic force microscope, by Park Systems (1 mentions)
Bx51m, by Olympus (1 mentions)
11 protocols using u lh100 3
1
Hoechst 33342 Staining for Nuclear Morphology
2
Aortic Histology Analysis in Mice
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Mice were sacrificed by CO2 inhalation and perfused with saline. Aortas from Pparafl/fl and PparaΔSMC mice were fixed in phosphate-buffered 4% formalin for 24 h and then embedded in a Tissue-Tek O.C.T. compound (4583, Sakura Finetek USA, Torrance, CA, USA). Frozen sections (7 μm) of the thoracic aorta from Pparafl/fl and PparaΔSMC mice were continuously collected from the proximal to the distal. Hematoxylin (RY-ICH001a, Roby, Beijing, China) and eosin (RY-ICH002a, Roby, Beijing, China) (H&E) staining, elastin staining by Weigher’s elastic staining method (G1592, Solarbio, Beijing, China), and Masson’s trichrome staining were routinely performed as previously described [26 (link)]. The images were obtained using an Olympus laser scanning microscope (U-LH100-3, Olympus Corporation, Tokyo, Japan).
3
Measuring Mitochondrial Superoxide in VSMCs
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Mitochondrial superoxide, an important source of reactive oxygen species (ROS) in VSMCs, was measured by staining with MitoSOX Red (Invitrogen, M36008, Waltham, MA, USA). The VSMCs (1 × 105) from PparaΔSMC and Pparafl/fl mice upon the Ang II treatment (1 μmol/L) for 24 h were loaded with MitoSOX Red (5 μM) for 10 min at 37 °C and then washed. The images were acquired using an Olympus laser scanning microscope (U-LH100-3, Olympus Corporation, Tokyo, Japan).
4
Histological Analysis of Kidney Damage
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After the rats had been sacrificed, the ligated kidneys were fixed in 4% paraformaldehyde for histological examination. Kidney tissue was embedded in paraffin and cut into 3-μm-thick sections. Standard sequential techniques were used to dewax the sections before haematoxylin-eosin (HE) and Masson’s trichrome (MT) staining. Damage was assessed as described in previous studies [11 (link), 12 (link)].
The kidney sections used for immunohistochemical detection were incubated with primary antibodies against α-smooth muscle actin (α-SMA) (1:1000, Cell Signaling Technology, Inc., USA) and E-cadherin (1:1000, Cell Signaling Technology, Inc., USA) according to the manufacturer’s protocols. After washing, the sections were further incubated with anti-rabbit secondary antibody (Abcam, Cambridge, USA) at a 1:5000 dilution. Finally, the sections were counterstained with Mayer’s haematoxylin and dehydrated for further observation. The Graphic context analysis software ImagePro plus 6.0 was used to analyse the immunohistochemistry pictures. Five fields were chosen under a × 200 microscope (OLYMPUS U-LH100-3) for each slice to record the positive staining for the average integral optical density.
The kidney sections used for immunohistochemical detection were incubated with primary antibodies against α-smooth muscle actin (α-SMA) (1:1000, Cell Signaling Technology, Inc., USA) and E-cadherin (1:1000, Cell Signaling Technology, Inc., USA) according to the manufacturer’s protocols. After washing, the sections were further incubated with anti-rabbit secondary antibody (Abcam, Cambridge, USA) at a 1:5000 dilution. Finally, the sections were counterstained with Mayer’s haematoxylin and dehydrated for further observation. The Graphic context analysis software ImagePro plus 6.0 was used to analyse the immunohistochemistry pictures. Five fields were chosen under a × 200 microscope (OLYMPUS U-LH100-3) for each slice to record the positive staining for the average integral optical density.
5
Histological Analysis of Organ Pathologies
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After embedding, sectioning, and dewaxing, the colon, liver, and kidney tissues were stained with hematoxylin and eosin (H&E). The pathological characteristics of the liver, kidney, and colon tissues were then scrutinized under a microscope (magnification × 20, U-LH100-3, Olympus, Tokyo, Japan). Finally, we performed an inflammation score concerning this literature (Cheng et al. 2008 (link); Rank et al. 1995 (link)).
6
Quantifying Cell Death by TUNEL Assay
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DNA fragmentation was detected with a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-nick end-labeling (TUNEL) assay kit (DeadEnd TM fluorometric TUNEL system; Promega, Madison, WI, USA) according to the manufacturer’s protocol. In brief, cells grown on collagen coated coverslips were fixed with a freshly prepared 4% paraformaldehyde for 25 min at 4°C. Then, cells were washed two times with ice-cold PBS and permeabilized with 0.2% Triton X-100 in PBS on ice for 5 min. The TdT and fluorescein-12-dUTP reactions were performed for 1 h at 37°C in a humidified box. TUNEL-positive cells were analyzed under a fluorescence microscope (U-LH 100–3, Olympus).
7
Apoptosis Detection Using TUNEL Assay
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DNA fragmentation was detected with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-nick end-labeling (TUNEL) assay kit (DeadEnd TM fluorometric TUNEL system, Promega) according to the manufacturer's protocol. In brief, cells grown on poly-L-lysine coated chamber slides were fixed with a freshly prepared 4% paraformaldehyde for 25 min at 4℃, washed 2 times with ice-cold PBS and permeabilized with 0.2% Triton X-100 in PBS on ice for 5 min. The TdT and fluorescein-12-dUTP reactions were performed for 1 h at 37℃ in a humidified box, and then mounted with propidium iodide (PI) to stain all cells for counterstaining. TUNEL- and PI-positive cells were imaged using a fluorescence microscope (U-LH 100-3, Olympus, Tokyo, Japan). Both TUNEL – and PI-positive cells were counted and the numbers of apoptotic cells (TUNEL-positive cells) were expressed as a percentage of the total number of cells (PI-positive cells).
8
Immunofluorescence Staining of PPARα and αSMA
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The frozen sections were permeabilized with 0.1% Triton X-100 (9002-93-1, Sigma, MA, UK) and blocked with 10% goat serum (ZLI-9021, ORIGINE, Beijing, China) for 30 min at room temperature. The sections were incubated with the following diluted primary antibodies overnight at 4 °C: PPARα (1:100 dilution, PA1-822A, Invitrogen, Waltham, MA, USA) and αSMA (1:200 dilution, ab2961, Abcam, Madison, WI, USA). The fluorescent secondary antibodies, goat anti-rabbit TRITC (ZF-0316, ORIGINE, Beijing, China) and goat anti-mouse FITC (ZF-0312, ORIGINE, Beijing, China), were mixed and added, and the slides were further incubated at 37 °C for 1 h. The sections were then incubated with 4′,6′-diamidino-2-phenylindole (DAPI) and mounted. The images were acquired using an Olympus laser scanning microscope (U-LH100-3, Olympus Corporation, TKY, JPN).
9
Apoptotic Cell Uptake by Macrophages
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Jurkat cells were irradiated under a 254 nm UV lamp for 15 minutes to induce apoptosis, followed by incubation under normal cell culture conditions for 2–3 hours. This method routinely yields more than 85% apoptotic cells (ACs) as previously described (42 (link)). The ACs were resuspended at a concentration of 2 × 107 cells/mL, then incubated for 2 minutes with PKH26 red fluorescent dye (2 μM/107 cells). Isolated BMDMs were plated in dishes, and PKH26-labeled ACs were incubated with the macrophages for 45 minutes at a 1:5 macrophage/AC ratio. After 45 minutes, macrophages were washed 3 times with PBS to remove unbound ACs, and then the macrophages were fixed with 4% formaldehyde for 20 minutes. Images were taken by fluorescence microscopy (Olympus model U-LH100-3), and the percentage of BMDMs labeled with PKH26-tagged apoptotic Jurkat cells was quantified.
10
Biofilm Formation Assay with Microscopy
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The assay of biofilm formation was performed as previously reported with some modifications (Sun et al. 2017 (link)). In brief, 50 μL aliquots of each culture were inoculated into 5 mL fresh LB medium (with or without aTc) contained in two different 6-well polystyrene culture plates (final OD600 of ~ 0.05), followed by incubating at 37 °C without shaking for 24 h. Then, the medium was discarded, and the wells were gently rinsed with 1X PBS to remove planktonic and loosely adhered cells. The adhered biofilm was quantified by staining the cells with 1% crystal violet solution for 30 min at room temperature. The excess crystal violet was removed by washing the wells three times with distilled water. Crystal violet bound to the adhered cells was solubilized in 1 mL of 95% ethanol and quantified by measuring absorbance at 540 nm and 620 nm. For biofilm assay using a differential interference microscope (Olympus U-LH100-3, Japan), the sterilized coverslips were added to the well after inoculation as described above. After incubation for 24 h, the coverslips were taken out and washed gently using PBS buffer (0.15 M, pH 7.2) to remove the planktonic cells. The biofilm formation of each tested group was captured using the microscope. All experiments were performed with three independent biological replicates.
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