ECs were plated to confluence under normal growth conditions and treated with L-NIO (5 mM) ± NTG (5 μM) overnight. ECs were then detached and sequentially labeled with primary anti-ICAM-1 antibody (Santa Cruz Biotechnology, USA) for 20 min, followed by FITC-conjugated DyLight 488 anti-mouse IgG (Vector labs. USA). Next, ECs were fixed with 1% PFA, detected by a Cell Lab Quanta SC flow cytometer (Beckman Coulter, CA), and analyzed by FlowJo software (Treestar Inc, CA).
Dylight 488 anti mouse igg
Manufactured by Vector Laboratories
Sourced in United States
DyLight 488 anti-mouse IgG is a fluorescently labeled secondary antibody produced in goat that specifically binds to the heavy and light chains of mouse immunoglobulin G (IgG). It is conjugated with the DyLight 488 fluorescent dye, which has an excitation maximum of 493 nm and an emission maximum of 518 nm.
Automatically generated - may contain errors
Lab products found in correlation
Cell lab quanta sc, by Beckman Coulter (1 mentions)
Chamber slide, by Merck Group (1 mentions)
Dylight 594 anti rabbit igg, by Vector Laboratories (1 mentions)
Tcs sp8, by Leica (1 mentions)
E cadherin, by Zymo Research (1 mentions)
Occludin, by Zymo Research (1 mentions)
Eclipse e400, by Nikon (1 mentions)
Digital sight ds qi1mc camera, by Nikon (1 mentions)
Alexa fluor 594 phalloidin, by BD (1 mentions)
Af231, by R&D Systems (1 mentions)
Tgf β2, by R&D Systems (1 mentions)
Irdye 680rd goat anti mouse igg, by LI COR (1 mentions)
Nutlin 3, by Cayman Chemical (1 mentions)
Irdye 800cw goat anti rabbit igg, by LI COR (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Fibroblast growth factor 2 (fgf2), by Thermo Fisher Scientific (1 mentions)
Tnf α, by R&D Systems (1 mentions)
7 protocols using dylight 488 anti mouse igg
1
ICAM-1 Expression on Endothelial Cells
2
Immunofluorescent Staining of EphA2 and Shp2
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Immunofluorescent staining was performed as described previously by us43 (link). Briefly, cells were plated into chamber slides (Millipore) and fixed in 4% paraformaldehyde, permeabilized, and incubated with mouse anti-EphA2 antibody (1 : 300 dilution) and rabbit anti-Shp2 antibody (1 : 100 dilution), followed by incubation with DyLight® 488 anti-mouse IgG (DI-2788, Vector Laboratories) and DyLight® 594 anti-Rabbit IgG (DI-1794, Vector Laboratories). Images were captured using an inverted confocal fluorescent microscope (LEICA TCS SP8). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI).
3
Immunofluorescence Imaging of Cell Junctions
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To examine tight and adherens junctions, polarized epithelial cells were fixed with 4 % paraformaldehyde for 20 min, permeabilized with 0.05 % Triton X-100 and washed three times with PBS. Cells were then incubated with rabbit mAb occludin (Zymed) and rabbit mAb E-cadherin (CSI) for 1 h at room temperature. Cells were washed and incubated for 25 min with secondary antibody DyLight 488 antimouse IgG (Vector), and cell nuclei were stained with DAPI. Cells were analyzed using a Nikon Eclipse E400 fluorescence microscope.
4
Immunofluorescence Analysis of Placental sFLT1
5
Monocyte Adhesion Assay with ICAM-1 Clustering
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ECs were grown to confluence on glass coverslips under normal growth conditions and treated with L-NIO (5 mM) ± [NTG (5 μM) or NTG-NL (5 μg/ml)] for 24 hr. Next, a monocyte adhesion assay was performed (as described earlier) and the U937 cell-EC cocultures fixed, permeabilized with 0.1% Triton X-100, blocked with 2% bovine serum albumin (BSA; Millipore, USA), and sequentially incubated with primary anti-ICAM-1 mouse antibody (Santa Cruz Biotechnology, USA) and secondary FITC-conjugated DyLight 488 anti-mouse IgG (Vector Labs, USA). To visualize actin microfilaments, U937 cell-EC cocultures were incubated with Alexa Fluor 594-Phalloidin (BD Biosciences, USA). Coverslips were mounted onto glass slides and fluorescence images (15 per condition) were taken using a Nikon Eclipse Ti microscope fitted with a Nikon Digital Sight DS-Qi1Mc camera. ICAM-1 clustering index was determined by measuring ICAM-1 fluorescence intensity (from n ≥ 10 images) at the U937-EC adhesion site and normalizing it to the average ‘background’ intensity measured from three neighboring EC cytoplasmic sites.
6
Dual Immunofluorescence Staining of Kidney Biopsies
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Snap-frozen, nonfixed kidney biopsies were double immunofluorescence stained with sFLT1/CD68 antibodies or sFLT-1/CD34 antibodies. Five-micrometer cryosections of kidney biopsies were cut and equilibrated in phosphate-buffered saline for 10 minutes at 37 °C, followed by incubation with sFlt-1 (1:200; AF231; R&D Systems), CD68 (FLEX Monoclonal Mouse Anti-Human CD68, Clone KP1, (Agilent), or FLEX Monoclonal Mouse Anti-Human CD34 Class II, Clone QBEnd 10, (Agilent) for 30 minutes at 37 °C. Slides were then rinsed with phosphate-buffered saline and incubated for 30 minutes with VectaFluor R.T.U. DyLight 594 Anti-Goat IgG or VectaFluor R.T.U. DyLight 488 Anti-Mouse IgG (Vector Laboratories), respectively. Slides were rinsed twice in phosphate-buffered saline and mounted.
7
Investigating EMT Signaling Pathways
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TGF-β2 and TNF-α were purchased from R&D Systems (Minneapolis, MN, USA). EGF and FGF-2 were obtained from Peprotech, Inc (Rocky Hill, NJ, USA). Nutlin-3 was purchased from Cayman Chemical (Ann Arbor, Michigan, USA). Antibodies against N-cadherin, Vimentin, Cytokeratin (Catalog #4545), and MDM2 were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibody against Fibronectin was from Abcam (Cambridge, MA, USA). Antibodies against β-actin and p53 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibodies of IRDye 800CW goat anti-rabbit IgG and IRDye 680RD goat anti-mouse IgG were obtained from Li-cor Biotechnology (Lincoln, NE, USA). Secondary antibodies of DyLight 549 goat anti-rabbit and DyLight 488 anti-mouse IgG were purchased from Vector Lab (Burlingame, CA, USA).
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