Boyden chamber assay, as previously described (21) . Twentyfour-well Boyden chambers with Matrigel-coated filters (8 µm pore size) were purchased from Becton-Dickinson (San Diego, CA, uSA). Hs578T and MDA-MB-231 cells were resuspended in culture media (5x10 4 cells/well) and then added to the Matrigel-coated upper compartment of invasion chambers in the presence or absence of 50 ng/ml STC-1 and/or 2 µg/ml IgG or STC-1 antibody. Fresh culture media with 5% FBS was added to the lower compartment of the invasion chamber. After a 24 or 48 h of incubation, the cells on the upper side of the filter were removed using cotton swabs. The underside of the filter was fixed in 100% methanol, washed in 1x PBS and stained using hematoxylin and eosin (H&E). Cells that had invaded through the matrigel were located on the underside of the filter. These cells were analyzed using a ScanScopexT apparatus (Aperio Technologies, Vista, CA, uSA). Statistical analysis. Statistical significance was determined using Student's t-test. Results are presented as means ± SEM. All quoted P-values were two-tailed and differences were considered statistically significant when the P-value was <0.05. Statistical analyses were performed using Microsoft Excel.
Scanscope xt apparatus
Manufactured by Leica
Sourced in United States
The Scanscope XT is a high-resolution digital slide scanning system designed for pathology applications. It is capable of scanning glass slides and producing high-quality digital images for further analysis and review.
Automatically generated - may contain errors
5 protocols using scanscope xt apparatus
1
Boyden Chamber Invasion Assay
2
Breast Cancer Cell Invasion and Migration Assays
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Boyden chambers with/without Matrigel-coated filters (8 µm pore size) from BD were used for cell invasion/migration assays. For invasion assays, breast cancer cells were resuspended in fresh media (5 × 104 cells/well) and loaded into the upper chamber coated with Matrigel. For migration assays, M2 cells were resuspended in fresh media (1 × 104 cells/well) containing 100 ng/mL chemokine (C-C motif) ligand 2 (CCL2) recombinant protein and placed into the upper chamber. Fresh medium containing 5% FBS was added to the lower chamber. Cells were incubated at 37 °C for 48 h. After incubation, cells remaining in the upper chamber were removed. Invaded/migrated cells located on the bottom surface of the filter were fixed with 100% methanol, washed with phosphate-buffered saline (PBS), stained with toluidine blue dye, and analyzed using a Scanscope XT apparatus (Aperio Technologies, Vista, CA, USA).
3
Immunohistochemistry Assay Protocol for Tumor Analysis
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For immunohistochemistry assays, paraffin-embedded primary tumors and lung tissue sections were deparaffinized in xylene, dehydrated in graded alcohol, and hydrated in water. Tissue sections (4 μm) were evaluated by H&E and Ki-67 staining with the appropriate positive and negative controls. TUNEL staining was performed using the ApopTag Peroxidase In Situ Apoptosis Detection kit (Millipore, CA, USA) according to the manufacturer’s instructions. Quantitative staining data for Ki-67 and TUNEL were obtained by counting four fields per slide. The slides were analyzed using a Scanscope XT apparatus (Aperio Technologies, CA, USA) [23 (link)].
4
Breast Cancer Cell Invasion Assay
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Matrigel-coated filter inserts (8 μm pore size) that fit into 24-well invasion chambers were obtained from Becton-Dickinson. Breast cancer cells to be tested for invasion were resuspended in culture medium (5 × 105 cells/well) and then added to the matrigel-coated upper compartment of the invasion chamber in the presence or absence of 20 μM ZER. Fresh culture media with 5% FBS were added to the lower compartment of the invasion chamber. The chambers were incubated at 37°C for 24 h. After incubation, the cells on the upper side of the filter were removed using cotton swabs, and the bottom filters were fixed in 100% methanol, washed in 1× PBS, and stained using toluidine blue dye. Breast cancer cells that invaded through the matrigel were located on the underside of the filter. These cells were photographed using a Scanscope XT apparatus (Aperio Technologies Inc., CA, USA). The invasion rates were calculated by averaging the total number of cells from four filters.
5
Transwell Invasion Assay with Matrigel
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The 24-well Boyden chambers with matrigel-coated filters (8-μm pore size) were from Becton-Dickinson (San Diego, CA, USA). Control and ACTA2 shRNA-transfected cells were resuspended in culture media (5 × 104 cells/well) and added to the matrigel-coated upper compartment of invasion chambers. Fresh culture media with 5% FBS was added to the lower compartment. After 48 h, cells on the upper side of the filter were removed using cotton swabs. The underside of the filter was fixed in 100% methanol, washed in PBS, and stained using hematoxylin and eosin (H&E). Cells that invaded the matrigel were analyzed using a Scanscope XT apparatus (Aperio Technologies, Vista, CA, USA).
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