Rat BDE-spTDEH10 or human HuCCT1 or CCLP1 cells were plated at an initial cell density of 5 × 105 cells per well in six-well culture dishes and then allowed to form a confluent monolayer. After a 24-hour period without serum, the cell monolayer was scratched with a sterile pipette tip (200 μL), washed with serum-free medium to remove floating and detached cells, and then photographed (time 0 hours) under the 10× objective of an Olympus 1X71 microscope (Olympus Corp., Center Valley, PA). Next, cells were pretreated with JTE-013 (10 μM) or dimethylsulfoxide (DMSO) for 1 hour, then treated with TCA (100 μM) or S1P (100 nM). After 48 hours, the wounded area was photographed as described above. Images acquired for each treatment group were further analyzed using IPLab 4.0. imaging software (Scanalytics, Inc., Rockville, MD).
1x71 microscope
Manufactured by Olympus
Sourced in Japan, Germany
The Olympus 1X71 is a high-quality inverted microscope. It features a sturdy frame, a dedicated optics system, and various illumination options to support a range of microscopy techniques.
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Lab products found in correlation
Ds fi1, by Nikon (3 mentions)
Image pro plus 6, by Media Cybernetics (2 mentions)
Cellsens standard software, by Olympus (2 mentions)
Image pro express software v 6, by Olympus (1 mentions)
Dcfh da, by Nanjing Jiancheng (1 mentions)
Gb11063 3, by Wuhan Servicebio Technology (1 mentions)
Gb23303, by Wuhan Servicebio Technology (1 mentions)
Rabbit anti mouse cd3 antibody, by Abcam (1 mentions)
Ix71 microscope, by Olympus (1 mentions)
Vectashield dapi mounting medium, by Vector Laboratories (1 mentions)
Cell f software, by Olympus (1 mentions)
Leica sp5 inverted confocal microscope, by Leica Microsystems (1 mentions)
Axiovert 200m microscope, by Zeiss (1 mentions)
Image pro plus software 6, by Media Cybernetics (1 mentions)
Matrigel assay, by BD (1 mentions)
Mmp assay kit, by Beyotime (1 mentions)
Alexa fluor 488 goat anti rat igg, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent with 4 6 diamidino 2 phenylindole, by Thermo Fisher Scientific (1 mentions)
Protocol hema 3 manual staining system, by Thermo Fisher Scientific (1 mentions)
Ckx41, by Olympus (1 mentions)
33 protocols using 1x71 microscope
1
Wound Healing Assay with BDE-spTDEH10 Cells
2
Quantifying Cellular Oxidative Stress
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Reactive Oxygen Species (ROS) are associated directly with any cellular disorder or dysfunction as a result of stress. ROS were tested in microbes grown under cold stress as a measure of disturbance in regular cell functions. For this purpose, the Bacillus strains, i.e., FZB42, CJCL2, and RJGP41 were cultured at 4 °C overnight and the cells were harvested by centrifugation at 96 h post-inoculation. The harvested cells were incubated for 30 min at 25 °C in 1.5 mL Eppendorf tubes containing a mixture of 10 Mm sodium phosphate buffer with pH 7.4 and dichloro-dihydro-fluoresein diacetate (DCFH-DA) (JianCheng Bioengineering, Nanjing, China) [39 ]. This dye can stain the sample containing ROS and green fluorescence was observed with the help of fluorescent microscopy using Olympus1X71 microscope and Image Pro express software v.6.2 (Olympus, Tokyo, Japan).
3
Histological Evaluation of Skin Regeneration
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For HE and Masson staining, 4-μm-thick paraffin-embedded sample slices were prepared. These experiments were performed by Servicebio (CHN). Images were taken using an Olympus1X71 microscope (Olympus, JPN). Image-Pro Plus 6 (Media Cybernetics, USA) was used to measure the thickness of the epidermis and evaluate the integrity of the new skin in the HE staining. Collagen was quantified by calculating the ratio of the positive area (blue–green collagen) to the total area in the skin tissue Masson staining.
In order to detect the presence of CD31, immunohistochemistry was carried out. Rehydrated paraffin sections were incubated in a microwave oven for 8 min. All sections were blocked with 3% BSA at 37 °C for 30 min, followed by incubation with anti-CD31 antibody (Servicebio, GB11063-3, 1:800) at 4 °C overnight. After washing with PBS, a goat anti-rabbit secondary antibody (Servicebio, GB23303, 1:200) coupled with horseradish peroxidase was employed. Samples were incubated at 37 °C for 1 h. Sections were visualized with diaminobenzidine tetrahydrochloride, and staining was detected with a light microscope (Olympus, JPN). CD31 levels were quantified by calculating the ratio of the positive area (yellowish-brown) to the total area using Image-Pro Plus 6 (Media Cybernetics, USA).
In order to detect the presence of CD31, immunohistochemistry was carried out. Rehydrated paraffin sections were incubated in a microwave oven for 8 min. All sections were blocked with 3% BSA at 37 °C for 30 min, followed by incubation with anti-CD31 antibody (Servicebio, GB11063-3, 1:800) at 4 °C overnight. After washing with PBS, a goat anti-rabbit secondary antibody (Servicebio, GB23303, 1:200) coupled with horseradish peroxidase was employed. Samples were incubated at 37 °C for 1 h. Sections were visualized with diaminobenzidine tetrahydrochloride, and staining was detected with a light microscope (Olympus, JPN). CD31 levels were quantified by calculating the ratio of the positive area (yellowish-brown) to the total area using Image-Pro Plus 6 (Media Cybernetics, USA).
4
Immunohistochemistry of Liver and Kidney Tissues
5
Osteogenic Differentiation Assay in MSCs
6
Immunofluorescence Staining of Fixed Cells
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Samples were fixed in 4% paraformaldehyde for 1 h or overnight at 4°C, permeabilized with 0.5% Tween in 1× PBS for 20 min, and blocked with 10% FBS diluted in 0.1% Tween in 1× PBS for 1 h. Primary antibodies were diluted at 1:500 in blocking solution, and samples were incubated overnight at 4°C rotating. Secondary antibodies were diluted at 1:300 in blocking solution, and samples were incubated for 1 h at room temperature, washed, and covered with 0.1% Tween in 1× PBS containing DAPI VectaShield mounting medium (Vector Laboratories). A list of the antibodies used is in Supplemental Table S7. Images were taken on either an Olympus 1X71 microscope with Cell^F software (Olympus Corporation), a Zeiss Axiovert 200M microscope with AxioVision release 4.7 software (Carl Zeiss Ltd.), or a Leica SP5 inverted confocal microscope (Leica Microsystems Ltd).
7
Matrigel Tube Formation Assay
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Tube formation of MMVECs was assessed using a Matrigel assay (BD Biosciences, Franklin Lakes, NJ, USA). Matrigel was chilled at 4°C overnight, melted prior to use and then quickly added (70 µl/well) to 96-well plates using a pre-chilled pipette. The plates were incubated at 37°C in 5% CO2 for 1 h. The cells were seeded into the plates at a density of 1×104 cells/well and incubated for 12 h in the presence or absence of IMD1–53 (80 nmol) and/or Comp C (20 µmol) at 37°C. Tube formation was observed and images were captured using an Olympus 1X71 microscope (Olympus Corporation). Images were processed using Image-Pro Plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) to calculate the degree of tube formation by measuring the length of tubes from five randomly selected fields (magnification, ×200) from each well. Each experiment was repeated three times.
8
Wound Healing Assay for Pancreatic Cancer Cells
9
Quantifying Germ Cell Apoptosis in Nematodes
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Germ cell apoptosis was assessed as previously described (Du et al., 2015) . Briefly, nematodes treated with SiO 2 NPs were stained with AO and then taken out and placed on NGM plates for 40 min to recover. Nematodes were immobilized by levamisole and observed under an Olympus 1X71 microscope (Olympus, Tokyo, Japan). Normal gland cells were green and apoptotic gonad cells were bright yellow or orange. Thirty nematodes in each group were examined.
10
MMP Evaluation in KGN Cells
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MMP was detected by MMP assay kit with JC-1 (Beyotime, China). KGN cells were incubated with 1 ml staining working solution (8 ml ultrapure water + 50 μl JC − 1 (200X) + 2 ml JC − 1 staining buffer (5X)) and 1 ml FBS-free medium per well (6-well plates) at 37°C in the dark for 20 min, then washed with JC-1 iced staining buffer (1X), and maintained in FBS-free medium while being examined.
All of the fluorescence intensity was examined under an Olympus 1X71 microscope and analyzed with ImageJ. The results are from 3 replications.
All of the fluorescence intensity was examined under an Olympus 1X71 microscope and analyzed with ImageJ. The results are from 3 replications.
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