Tissue samples were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Continuous sections were made with a thickness of 5 μm and a distance of 30 μm. Toluidine blue staining was performed after routine dewaxing, and images were captured under a Leica AM6000 microscope. The number of MCs was counted with Image J software.
Am6000
Manufactured by Leica
Sourced in Germany
The AM6000 is a high-performance microscope designed for advanced material analysis. It features a modular design and supports a wide range of imaging and analytical techniques, including scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). The AM6000 delivers reliable and accurate results for a variety of applications in the fields of materials science, nanotechnology, and industrial research.
Automatically generated - may contain errors
Lab products found in correlation
Crystal violet, by Merck Group (3 mentions)
Crystal violet, by Beyotime (2 mentions)
Matrigel, by BD (2 mentions)
Upper transwell chamber, by Corning (2 mentions)
Ab138515, by Abcam (1 mentions)
Citric acid, by Agilent Technologies (1 mentions)
Ab15580, by Abcam (1 mentions)
Upper transwell chamber, by BD (1 mentions)
Transwell plate, by Corning (1 mentions)
Matrigel coated chamber, by BD (1 mentions)
Transwell chamber, by BD (1 mentions)
Click it edu, by Thermo Fisher Scientific (1 mentions)
10 protocols using am6000
1
Quantifying Mast Cells in Tissue Sections
2
Wound Healing Assay of PM2.5-Exposed Cells
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The A549 and H1299 cells were treated with 50 μg/cm2 concentrations of PM2.5 for 72 h, and then PM2.5-exposed cells were plated at a density of 2 × 106 per well in six-well culture dish. The PM2.5-unexposed cells were seeded as control groups. Cells were then allowed to scratch approximately 300 μm width after 12 h with the 10 μl pipet tip (Gene Era Biotech, USA), at the end of which, The media and displaced cells were then removed, and cultured with 2% low serum culture medium. This procedure makes it possible to image the entire width of the wound using a 5 × objective (Leica AM6000, Germany). Photos were taken of the scratches, including the reference points, at 0, 24, and 48 h to monitor closure of the scratch. The migrated distance was calculated for 24 and 48 h based on the reduction of the scratch width with respect to the 0 h time point. Images are analyzed by digitally drawing lines (using Adobe Photoshop) averaging the position of the migrating cells at the wound edges.
3
Colony Formation Assay Protocol
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Cells with indicated treatment were trypsinized and resuspended in a culture medium. Cells were seeded into a six-well plate (2000 cells/well) and cultured for 14 days and the culture medium was changed every 3 days during the period. After 14 days, cells were fixed with 4% paraformaldehyde at room temperature for 10 min and stained 0.5% crystal violet (Beyotime, Shanghai, China) for 20 min. Subsequently, the number of colonies was counted and the morphology of the colonies was photographed under Leica AM6000 microscope.
4
Immunohistochemical Analysis of Tumor Tissues
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Tumor tissues were fixed with 4% paraformaldehyde (PFA, V/V), followed by paraffin embedding. 4-μm tissue sections in paraffin were soaked in xylene for 15 min before dehydration with gradient ethanol. Each section was then soaked with citric acid (pH 6.0 DAKO) for 10 min at 95° for antigen retrieval and cooled to ambient temperature. After washing with TBST buffer for a 15-min period, the sections were incubated with 3% H2O2 for 10 min. Tissue sections were blocked using 5% bovine serum albumin (BSA) for 30 min, followed by overnight incubation using primary antibodies E2F2 (Ab138515, Abcam) and Ki67 (ab15580, Abcam) under 4°C. Color development was performed using a DAB color-rendering kit (Soleibol, Japan). The images were captured using a Leica AM6000 microscope.
5
Transwell Assay for OS Cell Migration and Invasion
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The effect of DPY30 knockdown on OS cell migration and invasion was determined by a Transwell assay as previously described.18 (link) Briefly, OS cells were suspended in 100 μL of serumfree DMEM and seeded at the density of 2×104 cells/well in the upper chambers of Transwell plate (24-well plate, 8-mm pore size, Corning, NY, USA). The transwell upper chamber (Corning) without Matrigel (BD Biosciences, MA, USA) was used for migration assay, while transwell upper chamber coated with Matrigel was used for invasion assay. Then, 500 μL of DMEM containing 10% FBS was added to the lower chambers. After culturing for 24 h, cells were fixed with 4% paraformaldehyde at room temperature for 30 min and stained with 0.1% crystal violet (Sigma, Germany) for 20 min. The non-migrating cells in the upper chambers were removed with a cotton swab. Cells were photographed under Leica AM6000 microscope and the number of invading cells was recorded in five random microscopic fields.
6
Colony Formation Assay for Cancer Cells
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SK-N-BE, SH-SY5Y cells were seeded into a 6-well plate (1000 cells/well) and cultured for 14 days. The culture medium was changed every 3 days during the period. After 14 days, cells were fixed with 4% paraformaldehyde at room temperature for 10 mins and stained with 0.5% crystal violet (Sigma, 109,218) for 45 mins. After wash with PBS, the number of colonies was counted and photographed under Leica AM6000 microscope.
7
Ovarian Cancer Cell Migration and Invasion Assay
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The migration and invasion ability of the ovarian cancer cells was detected using a 24-well Transwell chamber (Costar; Corning, Inc.) with an 8.0-µm pore size. The transfected cells were suspended in serum-free DMEM and inoculated in the upper chamber at a density of 5.0×105 cells/well. Matrigel-coated chambers (BD Biosciences) were used for the invasion assay, while chambers without Matrigel coating were used for the migration assay. The chamber coating was performed at 37°C for 30 min. Medium containing 10% FBS was added to the lower chamber with or without rhCTGF. After 48 h of incubation at 37°C, the migratory or invading cells on the membrane were fixed with methanol and stained with 0.1% crystal violet (Beyotime Institute of Biotechnology) at ambient temperature for 20 min. The number of migrating and invading cells was counted in 5 random fields using a Leica AM6000 microscope.
8
Scratch Wound Assay for SKOV-3 and CAOV3 Cells
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Transfected SKOV-3 and CAOV3 cells with or without rhCTGF were seeded into 6-well plates at a density of 5.0×105 cells/well. Cells were serum-starved for 18 h. When the degree of confluence reached ~90%, a scratch wound was created in the cell layer using a sterile 200-µl pipette tip in the central region of each well. The wounded cells were incubated at 37°C for 48 h. Cell images were then captured using an inverted light microscope (Leica AM6000). The distance that the cells migrated was analyzed using ImageJ software (version 1.8.0) (National Institutes of Health). The migration rate was calculated as ratio of the wound distance at 48 h to the wound distance at 0 h.
9
Transwell Assay for Cell Invasion and Migration
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Cell invasion and migration were assessed by transwell assay. Briefly, cells with different treatments were trypsinized and resuspended in serum-free medium. The transwell upper chamber (Corning, New York, USA) without Matrigel (BD Biosciences, MA, USA) was used for migration assay, while transwell chamber coated with Matrigel was used for invasion assay. A total of 2.5 × 105 cells were seeded into the upper chamber in 400 μL serum-free medium, and 500 μL of 20% serum-containing medium was added to the lower chamber. After 24 hours, cells on the membrane were fixed with 3.7% paraformaldehyde for 10 min and stained with 0.5% crystal violet (Sigma, Germany) for 20 min. Cells were photographed under a Leica AM6000 microscope.
10
EdU Incorporation Assay for Cell Proliferation
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Cells (2 × 10 5 per well) were inoculated in the 24-well plates, and EDU staining kit (Click-iT® EdU, Invitrogen) was used for Edu incorporation assay. When cells reached 80%, the culture medium was replaced with the medium containing 1× EdU and incubated for 2 h. After rinsing by PBS, 4% formaldehyde was added to fix cells for a 15 min, followed by 20 min incubation with 0.5% Triton X-100 in PBS. Then, Click-iT reaction mixture (0.5 mL) added to the fixed cells for 30-min incubation. The stained cells were washed by PBS with 3% BSA, and counterstained with DAPI (1 g/mL) for 15 min in the dark environment. After staining and wash with PBS, the images were captured under Leica AM6000 microscope.
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