Cells were directly seeded on glass coverslips coated with poly-L-Lysine 0.01% (Sigma, St-Louis, MO, USA cat. P4832) after Lipofectamine 3000-mediated DNA transfection, according to the manufacturer’s instructions (ThermoFisher-Invitrogen, Waltham, MA, USA cat. L3000008). Twenty-four hours later, the cells were fixed with PFA (3.7%), then permeabilized with 0.1% Triton X-100 in PBS and then incubated with the indicated primary and secondary antibodies. The coverslips were stained with a DAPI-containing solution (ThermoFisher, Waltham, MA, USA cat. 62248) and mounted using the anti-quenching solution Fluormount G (Southern Biotech, Birmingham, AL, USA). Fluorescent confocal images were acquired on a Zeiss LSM 880 AiryScan confocal microscope. The pictures were analyzed with ImageJ software.
Dapi containing solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
DAPI-containing solution is a fluorescent stain used for labeling and visualizing DNA in biological samples. It is a widely used tool in various applications, such as flow cytometry, fluorescence microscopy, and cell biology research.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880 airyscan confocal microscope, by Zeiss (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Poly l lysine 0.01, by Merck Group (1 mentions)
Fluormount g, by Southern Biotech (1 mentions)
Donkey serum, by Bio-Rad (1 mentions)
Labtek removable chamber slides, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
2 protocols using dapi containing solution
1
Immunofluorescence Imaging of Transfected Cells
2
Immunofluorescence Staining of dCas9-Fusion Proteins
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For immunofluorescence staining, HEK-293T cells transiently transfected with the dCas9-fusion constructs were seeded onto Lab-Tek removable chamber slides (ThermoFisher, Cat. No. 154534 PK) 24 h after transfection and induced with Dox (50 ng/L) 8 h later. After 24 h of Dox induction, the cells were fixed using 4% paraformaldehyde (Sigma, Cat. No. 1.00496), permeabilized with 0.1% Triton X-100 (Sigma, Cat. No. T8787), and blocked with PBS buffer containing 5% donkey serum (Bio-Rad, Cat. No. C06SBZ). The slides were incubated at 4 • C overnight with mouse anti-Cas9 primary antibody (Cell Signaling Technology, Cat. No. 14697) diluted in blocking solution (1:500). After incubation, the slides were washed with blocking solution and incubated for 1 h at room temperature in the dark with donkey anti-mouse Alexa Fluor 594 (1:250; ThermoFisher, Cat. No. A-21203). Finally, the slides were washed with blocking solution and mounted with DAPI-containing solution (ThermoFisher, Cat. No. 00-4959-52). The cells were imaged with a SP8 Falcon system Leica microscope and the images were processed using ImageJ software.
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