Cells were fixed with 4% paraformaldehyde (m/v) in PBS and permeabilized with Triton™ X-100 0.3% (v/v) (Sigma-Aldrich). Anti-ROCK2 (sc-1851) (Santa Cruz Biotechnology. Inc, Dallas, USA) and exposed to anti-Vimentin (SAB4300676 Sigma-Aldrich) polyclonal antibodies as primary antibodies. In all cases, the final concentration of primary antibody was 0.4 μg/mL. As secondary antibodies, we used a Texas Red-labeled (sc-3923) or CFL-647 (sc-362292) at 0.15 μg/mL. For F-actin fiber staining, we incubated with 50 μg/mL of Faloidin conjugated with Fluorescein Isothiocyanate (FITC) (Sigma – Aldrich P5282) for 40 min at room temperature, and nuclei labeled with DAPI (4',6-diamidino-2-fenilindol) (Sigma-Aldrich). We obtained the images by using an inverted Nikon Eclipse Ti microscope. For quantifying the mean fluorescence intensities (MFI), we used the NIS-Elements program for n = 3 and an average of 100 cells per replicate and sample.
Dapi 4 6 diamidino 2 fenilindol
Manufactured by Merck Group
Sourced in United States
DAPI (4',6-diamidino-2-fenilindol) is a fluorescent dye used in molecular biology and microscopy applications. It binds strongly to DNA and emits blue fluorescence when excited by ultraviolet light. DAPI is a widely used stain for visualizing and identifying nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
Anti vimentin, by Merck Group (1 mentions)
Triton x 100 0.3, by Merck Group (1 mentions)
Rhodamine phalloidin, by Cytoskeleton (1 mentions)
Mitotracker deep red, by Thermo Fisher Scientific (1 mentions)
White light laser, by Leica (1 mentions)
Hc pl apo cs2 63 1.40 oil immersion objective, by Leica (1 mentions)
3 protocols using dapi 4 6 diamidino 2 fenilindol
1
Immunofluorescence Staining of ROCK2 and Vimentin
2
Cellular Uptake of Nanoparticles Imaged
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To confirm cellular uptake of nanoparticles, confocal laser scanning microscopy imaging was performed on HeLa and EA.hy926. Six hours after SFNs, IB-SFNs, and FA-IB-SFNs exposures, cells were washed with PBS 1x and fixed for 10 min in 4% paraformaldehyde. Actin filaments were stained for 30 min of incubation with rhodamine-phalloidin (Cytoskeleton, Inc., Denver, CO, USA) and DAPI (4′,6-diamidino-2-fenilindol) (Sigma-Aldrich, St. Louis, MO, USA). Confocal images were obtained with a Leica STELLARIS 8 inverted confocal laser scanning microscope, a white laser, and a FRET-FLIM module.
3
Immunofluorescent Imaging of Mitochondrial Sirt3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence analysis was performed as described previously (17) . Briefly, cells were labelled with 200 nM MitoTracker Deep Red (ThermoFisher Scientific, USA), fixed with 2% paraformaldehyde, incubated with anti-Sirt3 primary polyclonal antibody (dilution 1:100, Santa Cruz, USA), followed by FITC-labeled Goat anti-Mouse IgG secondary antibody (dilution 1:100, Proteintech, USA). DAPI (4,6-diamidino-2-fenilindol, Sigma Aldrich, St. Louis, MO, USA) was used for nuclear staining. Confocal imaging was performed by sequential scanning using Leica TCS SP8 X laser scanning microscope, equipped with a HC PL APO CS2 63×/1.40 oil immersion objective and a white light laser (Leica Microsystems, Germany). The excitation wavelengths and emission detection ranges used were 405 nm and 420-477 nm for DAPI, 490 nm and 500-600 nm for FITC, 644 nm and 665-780 nm for MitoTracker Deep Red.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!