MCF-7 and BT20 cells were seeded in a 96-well plate (Corning, USA) at a density of 8,000 cells/well and then treated with various concentrations (10, 32, 100 μM) of DMDD for 2 h, followed by incubation with 10 ng/ml tumor necrosis factor-alpha (TNF-α) (Sigma-Aldrich, USA) for 30 min. Untreated cells and cells treated with 10 ng/ml TNF-α alone served as controls. Cells were fixed, permeabilized, and sequentially stained with NF-κB p65 primary antibody (Cell Signaling Technology, USA), DyLight 488-conjugated secondary antibody, and Hoechst 33342 dye. The Hoechst and DyLight fluorophores detect changes in nuclear morphology (blue fluorescence) and NF-κB distribution (green fluorescence), respectively. The samples were analyzed on an Arrayscan VTI HCS Reader (Thermo Scientific, USA). The Nuclear Translocation BioApplication (Thermo Scientific, USA) was used for image acquisition and analysis. For each well, at least 400 cells were automatically acquired and analyzed. The translocation index was calculated by measuring the average intensity difference of NF-κB between the identified cytoplasmic region and nuclear region (MEAN_CircRingAvgIntenDiffCh2).
Dylight 488 conjugated secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
DyLight™ 488-conjugated secondary antibody is a fluorescent-labeled secondary antibody used for detection and visualization in various immunoassays and imaging applications. It is conjugated with the DyLight™ 488 fluorescent dye, which has excitation and emission maxima of 493 nm and 518 nm, respectively.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence microscope, by Olympus (3 mentions)
Arrayscan vti hcs reader, by Thermo Fisher Scientific (1 mentions)
Nf κb p65 primary antibody, by Cell Signaling Technology (1 mentions)
Tnf α, by Merck Group (1 mentions)
96 well plate, by Corning (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
4 protocols using dylight 488 conjugated secondary antibody
1
NF-κB Translocation Assay in MCF-7 and BT20 Cells
2
Quantitative Analysis of Ki-67 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transfected cells were fixed with 4% paraformaldehyde at room temperature for 20 min, followed by the addition of 0.05% Triton X-100 solution at room temperature for 10 min. After blocking with 3% BSA (Sigma-Aldrich; Merck KGaA) at 37°C for 90 min, cells were incubated with a primary antibody against Ki-67 (cat. no. 11882S; 1:1,000; Cell Signaling Technology, Inc.) at 4°C overnight, followed by probing with the DyLight™ 488-conjugated secondary antibody (cat. no. ab96899; 1:250; Abcam) at 37°C for 1.5 h in the dark. The nuclei were stained with DAPI (Roche Diagnostics) in the dark for 5 min at room temperature. Images were captured under a fluorescence microscope (Olympus Corporation; magnification, ×200) and the relative fluorescence intensity was used to quantify the Ki-67-postive cells in three randomly selected fields of view. The relative fluorescence intensity was normalized to the average optical density of the control group.
3
Immunofluorescence Staining of Ki67 in SKOV3 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The transfected SKOV3 cells were fixed with 4% paraformaldehyde at room temperature for 20 min, followed by the addition of 0.1% Triton X-100 solution at room temperature for 10 min. The cells were blocked with 3% BSA solution at 37˚C for 90 min. Subsequently, a primary antibody against Ki67 (cat. no. 11882S; 1:1,000; Cell Signaling Technology; Boston, MA, USA) was incubated with the cells at 4˚C overnight, followed by incubation with DyLight™ 488-conjugated secondary antibody (cat. no. 35553; 1:2,000; Invitrogen; Thermo Fisher Scientific, Inc.) at 37˚C for 1.5 h in the dark. Subsequently, DAPI was used to stain the cells for 5 min at room temperature. After mounting, the cells were observed under a fluorescence microscope (magnification, x200; Olympus Corporation).
4
Immunofluorescence Staining of Collagen I
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Treated cells were fixed in 4% paraformaldehyde for 20 min, followed by the addition of 0.05% Triton X-100 solution for 10 min. Cells were blocked with 5% normal goat serum and then incubated with a primary antibody (anti-Col I antibody, cat. no. 72026S). After incubation with DyLight™ 488-conjugated secondary antibody (Cell Signaling Technology, Boston, MA, USA), cells were stained with 4ʹ, 6-diamidino-2-phenylindole (Sigma-Aldrich; Merck KGaA). Images were captured under a fluorescence microscope (Olympus Corporation, Japan).
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!