Immunostaining was performed similar to previously described (Huffman, et al. 2007 (link); Huffman, et al. 2008 (link)). In brief, intestinal sections were subjected to antigen retrieval (Citrate buffer pH 6) using a pressure cooker on high steam for 10 min. Following rehydration, slides were treated with 3.0% H2O2 for 5 min to quench endogenous peroxidase activity, subjected to an avidin-biotin blocking step (Vector Labs SP-2001), and subsequently blocked with preimmune goat or rabbit serum (1%) for 20 min. Sections were then incubated with an antibody against Ki67 (1:400; cat#12202) pAktSer473 (1:50; cat#4060), phospho- (Ser/Thr) Akt Substrate (1:500; cat#9611), β-catenin (1:100; cat#8480), and anti-BrdU antibody (1:200; cat#5292) from Cell Signaling. A negative control was included in the same run using a subset of slides by omitting primary antibody from the staining procedure. Sections were then incubated with the appropriate biotinylated secondary antibody for 20 min, followed by a streptavidin-HRP detection system (Vector) and application of 3,3′-diaminobenzidine (DAB) for visualization of the antigen-antibody complex (Scytek). Digital files of all slides were then acquired with a PerkinElmer P250 High Capacity Slide Scanner and positive stained cells were analyzed using QuantCenter Software.
Anti brdu antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-BrdU antibody is a laboratory tool used to detect the presence of bromodeoxyuridine (BrdU) in cells. BrdU is a synthetic nucleoside that is incorporated into the DNA of dividing cells, allowing the identification of cells undergoing DNA replication. The Anti-BrdU antibody binds to BrdU, enabling the visualization and quantification of cell proliferation through techniques such as immunohistochemistry or flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Bromodeoxyuridine (brdu), by Merck Group (6 mentions)
Bromodeoxyuridine (brdu), by Cell Signaling Technology (2 mentions)
Brdu labeling reagent, by Thermo Fisher Scientific (2 mentions)
Sp 2001, by Vector Laboratories (1 mentions)
Phospho ser thr akt substrate, by Cell Signaling Technology (1 mentions)
P250 high capacity slide scanner, by PerkinElmer (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Aperio cs2, by Leica (1 mentions)
Dapi fluoromount g mounting medium, by Southern Biotech (1 mentions)
Ix71 inverted fluorescence microscope, by Olympus (1 mentions)
Thrombin, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Cell Signaling Technology (1 mentions)
5 bromo 2 deoxyuridine brdu, by BD (1 mentions)
Brdu cell proliferation chemiluminescent assay kit, by Cell Signaling Technology (1 mentions)
Brdu cell proliferation assay, by Cell Signaling Technology (1 mentions)
Propidium iodide solution, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Facscan, by BD (1 mentions)
Countess 3, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
25 protocols using anti brdu antibody
1
Immunohistochemical Analysis of Intestinal Tissue
2
Quantifying Cell Proliferation in Colon Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human colon cancer cells, siRNA transfected and grown on coverslips, were treated with OA and with BrdU (10 µM) (Sigma) for the final 6 h. Cells were fixed with 3.7% paraformaldehyde and stained with anti-BrdU antibody (Cell Signaling) and visualized using the slide scanner Aperio CS2 (Leica) and Image Scope software (independent experiments four times). BrdU positive cells were counted as a percentage of total cells per high power field.
3
Cell Proliferation Assay by BrdU
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were plated on 24-well plates at similar densities. Fresh media with different pHs were added to the cells. After 24 h, the cells were incubated with 10 μM BrdU for 1 h and stained with anti-BrdU antibody (Cell Signaling Technology) according to manufacturer's instructions. The cells were then incubated with anti-mouse Alexa Fluor594-conjugated secondary antibodies for flow cytometry analysis (Guava). The BrdU (#5292) antibody was purchased from Cell Signaling Technology.
4
Cell Proliferation Assay with BrdU Labeling
5
Cell Cycle Analysis by BrdU Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The frequency of cells in S-phase was determined by bromodeoxyuridine (BrdU) incorporation assay. First, 1 × 105 cells were seeded into 35 mm Petri dishes and samples were treated with 3a for 24 h at 5–10 μM. Two hours before the end of the treatment, a BrdU (CAS-number 59–14-3) pulse (100 µM) was performed. After that, BrdU immunodetection was carried out. Cells were fixed in 3.7% formaldehyde (15 min) at room temperature, and DNA was denatured using 1.5 mol L−1 of HCl (30 min). After washing, the samples were incubated with anti-BrdU antibody (Cell signaling, #5292, 1:500) overnight at 4 °C. Afterward, anti-mouse secondary antibody-FITC conjugated (1:100, Sigma) was added to the samples (2 h at room temperature), followed by DAPI staining (1 h at room temperature) (#D3571, ThermoFisher, Waltham, MA, USA). Cytological preparations were analyzed using a fluorescence microscope (Nikon, Tokyo, Japan, eclipse 80i).
6
Thrombin-Induced BrdU Incorporation in HFL1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HFL1 cells were treated with thrombin (1 U/ml, Sigma-Aldrich, St. Louis, MO) for 6 h and then labeled with 10 μM 5-bromo-2’-deoxyuridine (BrdU) (BD Pharmigen, San Jose, CA) for 18 h. Cells were stained for nuclei and BrdU using 4’,6-diamidino-2-phenylindole (DAPI) and anti-BrdU antibody (Cell Signaling, Danvers, MA) [36 (link)]. Results are expressed as the percentage of DAPI-stained cells that were also BrdU-positive.
7
BrdU Cell Proliferation Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Measurement of cell proliferation was achieved by using the BrdU Cell Proliferation Chemiluminescent Assay Kit (Cell Signalling Technology) following the supplier's instruction. Cells were seeded at a density of 5 × 104 cells/well and incubated for 24 h prior to exposure. Post exposure, the complete media was replaced with BrdU solution diluted in complete media (1/1000 dilution) and the cells returned to the incubator for 48 h to allow for BrdU incorporation. Subsequently, cells were fixed in 4% PFA for 15 min, washed in PBS and denatured (Cell Signalling Technology). After washing twice in PBS, anti-BrdU antibody (Cell Signalling Technology, 1/100 dilution) was added and incubated for 1 h at room temperature. Cells were then washed and blocked with 5% fetal calf serum for 45 min prior to adding anti-mouse 647 secondary antibody (Life Technologies, 1/400 dilution) and incubating for 1 h at room temperature. Finally, the cells were washed twice in PBS and imaged.
8
Viability and Proliferation Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The in vitro viability assay WST-1 [Cleavage of the tetrazolium salt WST-1 (4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) to formazan] was performed according to manufacturer’s instructions (Clontech, Mountain view, USA). Cells were plated at a density of 106 cells/ml and incubated for 24 hours (h) at 37 °C. Plates were read at 450 nm wavelength on a DAS plate reader (Rome, Italy). Results from WST-1 viability assays are expressed as fraction of treated versus untreated cells. Trypan blue exclusion, as well as Annexin V and propidium iodide (PI) methods were used to determine cell death. The BrdU Cell Proliferation Assay which detects 5-bromo-2′-deoxyuridine (BrdU) incorporated into cellular DNA during cell proliferation using an anti-BrdU antibody, was performed according to manufacturer’s instructions (Cell signaling Technology, Danvers, U.S). Cells were plated at a density of 106 cells/ml and incubated for 24 hours (h) at 37 °C. Plates were read at 450 nm wavelength on a DAS plate reader (Rome, Italy).
9
BrdU Incorporation and Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BrdU (Sigma) was added to the cell culture medium at a final concentration of 10 μM and incubated for 1 hour. After BrdU incorporation, cells were collected, fixed in 70% iced ethanol, and incubated at 4°C for 1 day. Cells were then treated with 0.5% Triton X‐100 (Sigma) for 30 minutes followed by centrifugation to remove the supernatant. The cells were resuspended in 0.1 M NaHCO3 for 30 minutes, centrifuged, and stained with anti‐BrdU antibody (Cell Signaling, Danvers, Massachusetts). Following staining with anti‐mouse FITC‐conjugated secondary antibody (Leinco Technologies, Baldwin, Missouri) for 30 minutes in the dark, the cells were then suspended in 10 μg/mL propidium iodide solution (Sigma) and analyzed by a flow cytometer (FACScan; Becton Dickinson, Franklin Lakes, New Jersey).
10
Evaluating U251MG Cell Proliferation
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!