Animals were sacrificed at 14 days post-operation. The thrombus together with surrounding venous wall was harvested. The specimens along the whole thrombus were fixed with 4% paraformaldehyde, embedded in paraffin, sliced 5 μm sections, and conducted with HE staining and immunofluorescent staining. Immunofluorescent staining against CD31, a sort of endothelial cells specific surface marker, was performed to illustrate the neovascularization in venous thrombus. The images of sections were taken by light microscopy (Nikon, Tokyo, Japan). The size of thrombus was quantified by measuring the area of each thrombus section using the image-analysis software (Image-Pro Plus, Media Cybernetics, Rockville, MD, UK). The newborn capillaries accounted for vascular recanalization were detected by counting the number of the CD31+ cells in 5 random fields under the light microscopy (Nikon, Tokyo, Japan).
Light microscopy
Manufactured by Nikon
Sourced in Japan
Light microscopy is a type of laboratory equipment used to observe and study small-scale objects and specimens. It utilizes visible light and a series of lenses to magnify and focus images of the subject, allowing for detailed examination and analysis.
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Lab products found in correlation
Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions)
Dab substrate, by Agilent Technologies (2 mentions)
In situ cell death detection kit, by Roche (2 mentions)
Hematoxylin and eosin, by Merck Group (2 mentions)
Rat anti mouse cd11b, by BD (1 mentions)
Mouse anti pcna, by Abcam (1 mentions)
Fitc conjugated goat anti rat igg, by Santa Cruz Biotechnology (1 mentions)
Fitc conjugated goat anti rat secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Confocal microscope, by Leica camera (1 mentions)
Live dead cell imaging kit, by Thermo Fisher Scientific (1 mentions)
Saturated picric acid, by Merck Group (1 mentions)
S 800 scanning electron microscope, by Hitachi (1 mentions)
Microtome, by Leica camera (1 mentions)
Gb11073 2, by Wuhan Servicebio Technology (1 mentions)
Ds u3 imaging system, by Nikon (1 mentions)
Safranin o fast green staining kit, by Solarbio (1 mentions)
Camcorder, by Nikon (1 mentions)
Ar advances researches, by Nikon (1 mentions)
H e staining kit, by Abcam (1 mentions)
96 protocols using light microscopy
1
Venous Thrombus Neovascularization Analysis
2
Hepatic Lipid Droplet Quantification
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Liver tissues were fixed with 4% paraformaldehyde solution for 24 h, a part of liver samples was dehydrated in alcohol, embedded in paraffin wax with a thickness of 4 mm section, followed by sliced at 5 μm, stained with HE using light microscopy (Nikon Corporation, Tokyo, Japan). Another part of liver tissue was dehydrated in sucrose solution, embedded in optimal cutting temperature compound, followed by being sliced at 8–10 μm and stained with ORO by light microscopy (Nikon Corporation, Tokyo, Japan) to identify the lipid droplets in the tissue. Lipid droplets are orange-red to bright red, and the nucleus are blue. The area of lipid droplets was quantified by Image-Pro Plus 6.0 software, and the percentage of lipid droplets area was calculated as the formula: % lipid droplets area = area of lipid droplets/area of liver tissue ×100.
3
Ferumoxytol Modulates Osteoclastogenesis
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Raw264.7 cells were seeded in 18 mm dishes with 2 × 104 cells per well. After overnight adherence, the medium was added with 50 ng/ml RANKL and different concentrations of Ferumoxytol. Then, the dishes were placed in geomagnetic field (GMF) as a control and 1–2 T SMF for 4 days, and the medium was changed every 48 h. Osteoclast formation was detected by TRAP staining Leukocyte Acid Phosphatase Kit (Sigma–Aldrich). F-actin Filament formation was examined by rhodamine-labeled phalloidin staining overnight at 4 °C.
Osteoclast bone resorption function was assessed by seeding Raw264.7 cells in 96-well Corning Osteo Assay Plate (Coring) with approximately 3000 cells per well. As similar with the previous exposure method, after 10 days of induction, the medium was removed and the cells were bleached in 10% sodium hypochlorite for 5 min at room temperature, and the wells were washed and dried. The resorption pits were observed by using light microscopy (Nikon) and analyzed by Image-J software.
Osteoclast bone resorption function was assessed by seeding Raw264.7 cells in 96-well Corning Osteo Assay Plate (Coring) with approximately 3000 cells per well. As similar with the previous exposure method, after 10 days of induction, the medium was removed and the cells were bleached in 10% sodium hypochlorite for 5 min at room temperature, and the wells were washed and dried. The resorption pits were observed by using light microscopy (Nikon) and analyzed by Image-J software.
4
Immunohistochemical Analysis of Spinal Cord and Microglia
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For immunohistochemistry staining, sections of spinal cords were incubated with rat anti-mouse CD11b (1:50, BD Pharmingen, San Diego, CA, USA), rabbit anti-mouse NG2 Ab (1:100, Chemicon International, USA & Canada), mouse anti-PCNA (1:5000 Abcam, Cambridge, UK,) or mouse anti-MBP (1:1000, Covance Inc., Princeton, New Jersey, USA), which were then labeled with FITC-conjugated goat anti-rat IgG (1:400, Santa Cruz, CA, USA), Cy3-conjugated goat anti-rabbit IgG (1:500, KPL, Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Maryland, USA), Alexa Fluor 488 goat anti-mouse IgG (1:500, Invitrogen) or HRP-conjugated goat anti-mouse IgG (1:1000, KPL), and examined by confocal microscopy (Leica Camera AG, Wetzlar, Germany) or light microscopy (Nikon). For immunocytochemistry staining, microglia were fixed in 4% paraformaldehyde for 20 min, blocked in 1% BSA for 30 min, and incubated overnight with rat anti-mouse CD11b (1:50, BD Pharmingen). Subsequently, cells were incubated with FITC-conjugated goat anti-rat secondary antibody (1:400, Santa Cruz). Finally, the cells were counterstained with DAPI and examined under a confocal microscope (Leica).
5
Live/Dead Cell Imaging Assay
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Live and dead cells were visualized by Live/Dead Cell Imaging kit (Life Technologies Corp., Eugene, OR) based on a cell permeable dye for staining of live cells (green) and a cell impermeable dye for staining of the dead cells (red). Cells were seeded in a 24-well plate overnight, treated with lapatinib for 24 hr, and freshly stained with Live/Dead Imaging kit exactly following the manufacturer's instructions. Cell images were subsequently acquired under a Nikon light microscopy.
6
Renal Fibrosis Assessment in Mice
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Paraffin sections of mouse kidneys were examined for renal fibrosis with the Picro-Sirius Red staining. Briefly, deparaffinized sections were treated with 0.1% Sirius Red in saturated picric acid (Sigma, St Louis, MO) and destained in 0.5% acetic acid. Collagen fibrils were stained and evaluated under light microscopy (Nikon) equipped with a polarizer. Ten randomly selected fields (magnification, x400) from cortex and medullar, respectively, in each kidney were evaluated and all images were captured by Olympus DP72 Capture Interface software. Ratio of Sirius red positive areas to whole areas in each field was calculated in percentages by Image J (NIH) software.
7
Migratory Potential of Cancer Cells
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The proliferative wound-healing assay was carried out to evidence the migratory properties of SGC7901/Tax and BGC823/Tax cells. Briefly, si-NC- or si-circ_002136-transfected cells were seeded into six-well plates for stable growth, followed by scratching when the cells reached 90–95% confluence. In monolayers of cells, pipette tips (200 μL, labeled 0 h) were introduced to create streaks followed by rinsing cells with phosphate buffered saline (PBS) and infusing fresh medium. After continuing incubation for 24 h, the rate of wound area closure was analyzed by light microscopy (Nikon, Tokyo, Japan, magnification ×40) and ImageJ (NIH).
8
Hyphal Morphology and Conidiation Analysis
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To analyze hyphal morphology, 7-day-old mycelial plugs (5 mm in diameter) were transferred onto fresh PDA plates, and then incubated at 25°C in dark conditions. Hyphae picked from edge and center of the colony on PDA were examined by light microscopy (Nikon, Tokyo, Japan). The extent of radial hyphal growth was measured after 7 days.
For conidiation, strains were cultured on PDA plates at 25°C in dark conditions for 7 days. Conidia were harvested from 9 cm diameter Petri dishes by suspending in 5 ml sterile distilled water per disc, and centrifuging at 3000 × g for 2 min. The solution was decanted, and then were counted with a hemocytometer under a light microscope.
For conidiation, strains were cultured on PDA plates at 25°C in dark conditions for 7 days. Conidia were harvested from 9 cm diameter Petri dishes by suspending in 5 ml sterile distilled water per disc, and centrifuging at 3000 × g for 2 min. The solution was decanted, and then were counted with a hemocytometer under a light microscope.
9
Evaluating Knee Osteoarthritis via Masson Staining
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Knee joint bone and cartilage specimens were decalcified with EDTA (Merck, Germany) embedded in paraffin wax (KUNHUA, China), and cut into 5 μm sections onto glass slides. Sealed with neutral gum after staining with Masson histochemical stain. Light microscopy (Nikon, Japan) was used to observe the specimen according to the manufacturer’s protocol. The Mankin histological score was also used to diagnose and score the knee osteoarthritis (KOA) [24 (link)].
10
Histological Analysis of Liver Tissue
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Liver tissues were fixed in 4% paraformaldehyde for 12–24 hr, dehydrated in absolute ethanol, transparentized in dimethylbenzene, and embedded in paraffin. Sections of 4 µm were cut, mounted on glass slides, deparaffinized, dehydrated with gradient ethanol, and routinely stained with hematoxylin–eosin (HE), and sealed with optical resin. The stained sections were observed by light microscopy (Nikon).
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