In order to understand the consequence of oxidative stress and to validate our findings, U937 cells were treated with a high concentration of exogenous H2O2 (5 mM) and incubated in standard conditions for 1 h. Subsequently, the samples were centrifuged at 850 × g for 10 min at 20°C, pellet was rinsed with phosphate buffer saline (PBS) and stained with acridine orange and propidium iodide (AO, 1.5 μM; PI, 1.5 μM). This double staining is a cell viability procedure that causes viable nucleated cells to fluoresce green and non-viable nucleated cells to fluoresce red. Acridine orange permeates viable cells and binds to nucleic acids. PI also binds to nucleic acids, but it is not able to permeate intact cell membranes, therefore is taken up only by non-viable cells and cells with compromised membranes. The maximum excitation/emission wavelengths are 500/526 nm for AO and 533/617 nm for PI. Fluorescence measurements were then performed using Olympus BX51TF (Olympus Corporation, Tokyo, Japan).
Bx51tf
Manufactured by Olympus
Sourced in Japan, United States, Belgium
The BX51TF is a research microscope designed for transmitted light applications. It features a sturdy, ergonomic design and a wide range of optical components to support various observation techniques, including brightfield, darkfield, and phase contrast microscopy.
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Lab products found in correlation
Image pro plus 6, by Media Cybernetics (3 mentions)
Mayer s hematoxylin solution, by Merck Group (2 mentions)
Image pro plus software, by Media Cybernetics (2 mentions)
Dp72 camera, by Olympus (2 mentions)
Mouse anti p63, by Abcam (2 mentions)
Fluoroshield histology mounting medium, by Merck Group (2 mentions)
Antigen retrieval solution, by Wuhan Boster Biological Technology (1 mentions)
Claudin 5, by Santa Cruz Biotechnology (1 mentions)
Claudin 4, by Santa Cruz Biotechnology (1 mentions)
Xl 30 esem feg scanning electron microscope, by Thermo Fisher Scientific (1 mentions)
Moticam 1080, by Motic (1 mentions)
Smz 171 tl, by Motic (1 mentions)
Anti cd20, by Agilent Technologies (1 mentions)
Anti mitf clone d5, by Agilent Technologies (1 mentions)
Matlab r2016a, by MathWorks (1 mentions)
Image pro plus med 6, by Motic (1 mentions)
Proliferating cell nuclear antigen (pcna), by Cell Signaling Technology (1 mentions)
Antifade mounting medium, by Beyotime (1 mentions)
Image pro plus, by Media Cybernetics (1 mentions)
Tcs spe, by Leica (1 mentions)
104 protocols using bx51tf
1
Oxidative Stress and Cell Viability Assay
2
Immunohistochemical Analysis of Dentin Matrix Proteins
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Subsequent to deparaffinization, the sections were treated with compound enzyme digestive juice (0.125% trypsin + 0.1% pepsin + 0.01% EDTA; Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 20 min and then with antigen retrieval solution (Wuhan Boster Biological Technology, Ltd.) for 10 min. The immunohistochemistry was performed according to the instructions of UltraSensitive™ SP (mouse/rabbit) immunohistochemistry kit (Maixin-Bio, Fuzhou, China). The anti-rat mouse monoclonal DMP1-C-8 G 10.3 and anti-rat mouse monoclonal anti-DSP-2C12.3 antibodies (donated by Dr Chunlin Qin, Baylor College of Dentistry, Texas A&M University Health Science Center, Dallas, TX, USA) (13 (link),14 (link)) at a dilution of 1:1,000 used as the primary antibodies (15 (link)). The sections were then observed under a microscope (Olympus BX51TF; Olympus Corporation).
3
Immunohistochemical Analysis of Claudin Expression in Rat Brain
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Immunohistochemistry was performed in normoxia- or hypoxia-exposed rat brain tissue as described by Beytut et al. [34 (link)]. Thin sections of the brain were taken and fixed with paraformaldehyde. Endogenous peroxidase activity was blocked with hydrogen peroxide (3 %) in distilled water for 30 min. The sections were incubated with PBS, (pH 7.2) for 5 min and subsequently placed into 0.05 % Trypsin EDTA for 20 min for antigen retrieval. After washing with PBS, the sections were incubated with 5 % normal goat serum for 60 min at RT. The sections were then incubated with each of the primary antibodies (claudin 4 and claudin 5 from Santa Cruz Biotechnology, Santa Cruz, CA, USA) for overnight at 4 °C. Following 4–5 times washing with PBST, the sections were incubated with HRP conjugated goat anti-rabbit IgG in PBST for 60 min at RT. Secondary antibodies were supplied by Sigma-Aldrich, St. Louis, MO, USA. Labelling was visualized with 3,3′-diaminobenzidine (DAB) as the chromogen. The images were captured by using Olympus BX51TF (Olympus Corporation, Centre Valley, PA, USA).
4
Fungal Conidia Germination Assay
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After treatments, conidia were centrifuged at 2000 g for 5 min, washed three times and resuspended with PD to a final concentration of 10 6 mL -1 in microwell plates. Germination was monitored for 36 h, using optical microscope Olympus BX51TF (Olympus Co., Tokyo, Japan). Conidia were considered germinated when germination tubes were at least twice as long as conidia diameters (Xiaoping et al. 2007) . Germination percentage was calculated from the obtained values.
5
Adhesion Analysis of BMSCs on Scaffolds
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In order to investigate the adhesion of BMSCs in different groups, cells were seeded in two different samples of each group at a density of 5 × 104 cells/ml. After 12 h of incubation, the samples were transferred to a new plate and smoothly washed 3 times with PBS and then fixed with 4% paraformaldehyde for 30 min at 4°C. Fixed samples were washed again with PBS for 2 min. The cell nuclei were stained with 4ʹ,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, USA) for 5 min and were observed by a scanning fluorescence microscope (Olympus BX51TF, Japan). In order to directly observe the morphology of the cells on the scaffold, cells were cultured on different samples at a density of 1 × 104 cells/ml for 3 days. Then, the samples with cells were rinsed 3 times with PBS for 2 min, fixed with 2.5% v/v glutaraldehyde at 4°C for 8 h, and dehydrated through an ethanol series. The samples were sputtered with Au before SEM observation (XL-30 ESEM FEG Scanning Electron Microscope, FEI Company).
6
Hippocampal BDNF and p-Akt Visualization
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Immunohistochemistry (IHC) was performed to visualize BDNF and p-Akt according to the manufacturer's instructions (Invitrogen). Briefly, free-floating 25 μm thick serial hippocampus sections were treated with 0.3% hydrogen peroxide for 10 min, and then, sections were rinsed three times with PBS and blocked in Reagent 1A for 10 min followed by incubation with BDNF (1 : 300) or p-Akt (1 : 500) antibody at 4°C overnight. After PBS washing, sections incubated with a biotinylated second antibody Reagent 1b followed by the conjugate enzyme Reagent 2 for each 10 min. Finally, a chromogen AEC single solution was utilized to develop the signals and captured in a microscope (BX51TF, Olympus, Tokyo, Japan) with cellSens standard V3 detection system.
7
Histopathological Analysis of Kidney Tissues
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H&E staining of paraffin-embedded kidney tissues of the index patient: 5 µm sections of paraffin-embedded tissues were mounted on super frost/plus glass and incubated at 60°C for 40 minutes. After deparaffinization, slides were incubated in Mayer's Hematoxylin solution (Sigma-Aldrich) and incubated with 1% Hydrochloric acid in 70% ethanol for 1 minute. Slides were then incubated for 10 seconds in Eosin (Sigma-Aldrich). Images were produced using Olympus BX51TF.
8
Automated Whole Tissue Imaging
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Virtual images were acquired with the fully automated digital microscopy system dotSlide (Olympus, BX51TF, Aartselaar, Belgium) coupled with a Peltier-cooled high-resolution digital color camera (1376×1032 pixels) (Olympus, XC10, Aartselaar, Belgium). Digital images of the whole tissue sections were digitized at high magnification (100×), producing virtual images with a pixel size of 1.510 µm.
9
Histological Analysis of Reproductive Organs
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Paraffin-embedded ovaries and uteri were sectioned serially at 4 µm with a microtome and were dried using a chip drying device after the sections had been adhered to slides. The slices from
different depths were stained with hematoxylin and eosin after deparaffinization and rehydration. After staining, the slices were sealed with neutral resin and mounted with cover slips.
Histomorphology was assessed using a virtual light microscope (model BX51TF; Olympus, Tokyo, Japan). The cross-sectional areas of the uterine cavity and uterine wall were established by
evaluating 15 slices (3 slices/uterus × 5 uteri) in each group. The structural characteristics of the ovary and uterus were observed from at least five mice in each treatment group.
different depths were stained with hematoxylin and eosin after deparaffinization and rehydration. After staining, the slices were sealed with neutral resin and mounted with cover slips.
Histomorphology was assessed using a virtual light microscope (model BX51TF; Olympus, Tokyo, Japan). The cross-sectional areas of the uterine cavity and uterine wall were established by
evaluating 15 slices (3 slices/uterus × 5 uteri) in each group. The structural characteristics of the ovary and uterus were observed from at least five mice in each treatment group.
10
Histological Analysis of Prostate Biopsies
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Standard surgical procedures for taking biopsies were employed. Prostate biopsies were immediately fixed in 10% buffered formaldehyde solution. Tissues were histologically processed using standard protocols. Three micrometer sectioned slides of prostate were hematoxylin and eosin stained and evaluated microscopically by competent pathologists for histological changes using an Olympus BX 51TF (Olympus Corporation; Tokyo, Japan) light microscope and reported accordingly as requested by the urologist. Patients proven positive histologically and willing to be part of the study were recruited.
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