Expanded monolayers were fixed after 7 days with 10% buffered formalin. Fixed monolayers were permeabilized using 0.2% Triton X-100 (Sigma-Aldrich) in PBS for 20 min, followed by incubation with phalloidin (TRITC-conjugated; 1/200 dilution in PBS; Sigma-Aldrich) for 1 h. Nuclei were stained with 100 ng/mL 4’,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) in PBS for 5 min. Monolayers were imaged using an upright fluorescent microscope (BX51; Olympus).
Tritc conjugated
Manufactured by Merck Group
Sourced in Italy
TRITC-conjugated is a fluorescent dye that is conjugated to various biomolecules, such as proteins, antibodies, or DNA. It has an excitation wavelength of around 550 nm and an emission wavelength of approximately 570 nm, which corresponds to the orange-red region of the visible spectrum.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
Triton x 100, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Clone v 9, by Merck Group (1 mentions)
Alexa633 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Mab240, by R&D Systems (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Digital sight u2, by Nikon (1 mentions)
Eclipse te300, by Nikon (1 mentions)
5 protocols using tritc conjugated
1
Fluorescent Imaging of Cytoskeletal Structures
2
Immunolabeling of Tissue Samples
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Prior to immunolabeling, tissue preservation, characterization and orientation were recorded by hematoxylin-eosin and Van Gieson stainings followed by histopathological analysis according to the EHRA classification [6] (link). Frozen sections were fixed for 10 min with 4 % paraformaldehyde. After washing in phosphate buffered saline (PBS) sections were incubated with 1 % bovine serum albumin for 30 min to block non-specific binding sites and then incubated with the primary antibodies. A monoclonal antibody against vimentin (clone V-9, Sigma) was directly conjugated with Cy3 (clone V9, Sigma) and a monoclonal antibody against TGF-β1 (MAB240, R&D) was used in dilutions as previously described [24] (link). Polyclonal primary antibodies against collagen V were purchased from Rockland. Anti-mouse or anti-rabbit IgG-conjugated with Cy3 or Cy2 (Biotrend) served as detection systems in single or double immunolabelings. The nuclei were stained with 1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI, Molecular Probes). F-actin was fluorescently stained using TRITC-conjugated (Sigma) or Alexa633-conjugated phalloidin (Molecular Probes). Negative controls were obtained by omitting the primary antibody, in an otherwise similar protocol. Sections were embedded in Mowiol and coverslipped.
3
Immunofluorescence Analysis of Nanoparticles in Endothelial Cells
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Endothelial cells (5x104 cells/well on glass cover-slips placed into 24 multiwell plates) were maintained in 10% FBS for 24 h. Then cells were treated with NPs or lyophilized gels (0.25 mg/ml and 0.5 mg/ml, respectively, 24 h) and fixed in acetone for 5 min. After blocking of unspecific bindings with 3% bovine serum albumin (BSA), cells were incubated overnight at 4°C with the primary antibody (anti β-actin, 1:70, Sigma). Samples were then incubated with a secondary antibody TRITC conjugated (Sigma) and analyzed by confocal microscopy (Zeiss LSM 700) at 60X magnification [21 (link)].
4
Actin and Nuclear Staining of Cultured Cells
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Cells were cultured on different surfaces for 72 hours, fixed in phosphate-buffered saline (PBS)—buffered formalin for 30 minutes and then labeled with phalloidin TRITC-conjugated (Sigma, Italy) to visualize actin filaments. 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was used for nuclear staining. Cells were observed by fluorescent microscope (Leica, TGS 4D) at a ×40 magnification.
5
Visualization of EGFR and phospho-EGFR in tumor cells
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Tumour cells (5 x 10 4 cells/well on glass cover-slips placed into 24 multiwell plates) were maintained in 10 % FBS for 48 hrs. Cells were fixed in acetone for 5 min. After the blocking of unspecific bindings in 3% BSA, cells were incubated overnight at 4°C with the primary antibody, an alexafluor 488 conjugate anti-rabbit EGFR (Cell signalling) or anti-rabbit phospho-EGFR (Y1045) (Cell signalling). Cells were then incubated with secondary antibody TRITC conjugated (Sigma Aldrich) and visualized by fluorescence microscope (Eclipse TE300, Nikon) at 60 X magnification (Nikon Plan Apo Ve, 60 X 1.4 Oil) and images taken by a digital camera (Nikon digital sight u2).
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