Cells cultured on coverslips were fixed in 4% paraformaldehyde in 1X PBS for 10 min at room temperature. After washing in PBS, the cells were incubated with 1X PBST (0.2% Triton X-100 in 1X PBS) for 20 min and then incubated in 5% FBS blocking solution for 30 min. The cells were incubated with α-tubulin and TPX2 antibodies at 4 °C overnight. After washing with 1X PBST three times for 5 min each, the coverslips were incubated with secondary antibody (Alexa Fluor-488 or 568 -conjugated IgG; (Invitrogen, Carlsbad, CA, USA) and DAPI (2.5 μg/ml) for 60 min and rewashed in 1X PBST three times for 5 min. The cells were mounted in 90% glycerol in Tris buffer (pH 10.0) and observed with a fluorescence microscope.
Alexa fluor 488 or 568 conjugated igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Alexa Fluor-488 or 568-conjugated IgG is a fluorescently labeled secondary antibody used for detection and visualization in various immunoassays and imaging applications. The Alexa Fluor dyes provide bright, photostable fluorescence signals that can be detected using standard fluorescence detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Ab11713, by Abcam (2 mentions)
Mab4841, by R&D Systems (2 mentions)
P enos, by Cell Signaling Technology (2 mentions)
Slowfade gold antifade mount with dapi, by Thermo Fisher Scientific (2 mentions)
Ls b5627, by LifeSpan BioSciences (2 mentions)
Anti α smooth muscle actin, by Thermo Fisher Scientific (2 mentions)
Mca1957, by Bio-Rad (2 mentions)
Anti cd31, by Santa Cruz Biotechnology (1 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
4 protocols using alexa fluor 488 or 568 conjugated igg
1
Immunofluorescence Labeling of Cytoskeletal Proteins
2
Cardiac Tissue Immunofluorescence Staining
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Hearts were fixed with 4% paraformaldehyde overnight at 4°C and embedded in paraffin. Serial sections (6 μmol L 1 ) were stained then incubated with 1 : 100 dilution of anti-CD31(Santa Cruz), or 1 : 200 dilution of anti-vimentin and 1 : 50 anti--smooth muscle actin (Epitomics) antibodies overnight at 4°C and then with a 1 : 100 dilution of Alexa Fluor 488 or 568-conjugated IgG (Invitrogen) for 1 h at room temperature. The images were captured with a TCS SP5 confocal microscope (Leica, Germany).
3
Immunohistochemical Characterization of Vascular Endothelium
4
Immunohistochemistry of Vascular Markers
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Tissue sections were deparaffined using xylene and rehydrated in a graded series of alcohols. Sections were heated in citric acid buffer (pH 6.0) at 100°C for 10 minutes for antigen retrieval. The sections were then blocked with 2% bovine serum albumin in phosphate-buffered saline (PBS) containing 0.1% Tween20, for 1 hour at room temperature, before incubation overnight at 4°C with the primary antibodies diluted in 2% bovine serum albumin in PBS. Primary antibodies used were anti-vWF (1:400, ab11713; Abcam, Cambridge, Mass), anti-Krüppel-like factor 2 (Klf2, 1:100, LS-B5627, LifeSpan Biosciences, Seattle, Wash), anti-phosphorylated endothelial nitric oxide synthase (p-eNOS, 1:50, 9570; Cell Signaling Technology, Danvers, Mass), anti-α-smooth muscle actin (1:500, 14-9760-82; eBioscience, San Diego, Calif), anti-CD68 (1:100, MCA1957, Bio-Rad, Hercules, Calif), and anti-CD3 (1:20, MAB4841, R&D, Minneapolis, Minn). Sections were then treated with secondary antibodies at room temperature for 1 hour using Alexa Fluor 488- or 568-conjugated IgG (Life Technologies, Eugene, Ore). Sections were stained with Slow Fade Gold Antifade Mount with DAPI (Life Technologies) and a coverslip was applied. Digital fluorescence images were captured and the intensity of the immunoreactive signal was measured using Image J software.
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