A549 and SPC-A-1 cells were seeded on sterile cover glasses placed in the 6-well plates. After transfection with miR-326 mimic, miR mimic NC, miR-326 inhibitor, miR inhibitor NC for forty eight hours, the BrdU (5-bromo-2-deoxyuridine; Sigma) stock solution at 10 mg/mL in saline was diluted 1000 × in the culture medium and incubated for 60 min. After washing with 1 × PBS, cells were then fixed for 20 min in 4% paraformaldehyde (PFA) and permeabilized with 0.3% Triton X-100 for 10 min. After blocking with 10% goat serum in 1 × PBS for 1 h, cells were incubated with a primary rabbit antibody against BrdU (1:200, Abcam) over night at 4°C, and then incubated with the secondary antibody coupled to a fluorescent marker, Cy3, at room temperature for 2 h. After DAPI staining and 1 × PBS washing, the cover slips were mounted on to glass slides with anti-fade solution and visualized using a fluorescence microscope (Olympus 600 auto-biochemical analyzer, Tokyo, Japan) with Image-Pro Plus software for image analysis, and 10 microscopic fields were taken for calculating BrdU.
Primary rabbit antibody against brdu
Manufactured by Abcam
Sourced in Japan
The primary rabbit antibody against BrdU (Bromodeoxyuridine) is a laboratory reagent used to detect and quantify cell proliferation. It specifically binds to BrdU, a synthetic nucleoside that is incorporated into the newly synthesized DNA of proliferating cells, allowing for the visualization and analysis of dividing cells.
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8 protocols using primary rabbit antibody against brdu
1
Evaluating Cell Proliferation with BrdU
2
BrdU Incorporation Assay for Cell Proliferation
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A549 and H1299 cells were seeded on sterile cover glasses placed in the 6-well plates. After transfection with miR-377-3p mimic or miR mimic NC, miR-377-3p mimic+E2F3, SiRNA NEAT1 or scrambled, NEAT1 or NC, for forty eight hours, the BrdU (5-bromo-2-deoxyuridine; Sigma) stock solution at 10 mg/mL in saline was diluted 1000 × in the culture medium and incubated for 60 min. After washing with PBS, cells were then fixed for 20 min in 4% paraformaldehyde (PFA) and permeabilized with 0.3% Triton X-100 for 10 min. After blocking with 10% goat serum in PBS for 1 h, cells were incubated with a primary rabbit antibody against BrdU (1:200, Abcam) over night at 4°C, and then incubated with the secondary antibody coupled to a fluorescent marker, Cy3, at room temperature for 2 h. After DAPI staining and PBS washing, the cover slips were mounted on to glass slides with anti-fade solution and visualized using a fluorescence microscope (Olympus 600 auto-biochemical analyzer, Tokyo, Japan) with Image-Pro Plus software for image analysis, and 10 microscopic fields were taken for calculating BrdU.
3
Measuring Cell Proliferation via BrdU Assay
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A549 and SK-MES-1 cells were seeded on sterile cover glasses placed in the 6-well plates. After transfection with miR-139-5p mimic, miR mimic NC, miR-139-5p inhibitor, miR inhibitor NC for forty eight hours, the BrdU (5-bromo-2-deoxyuridine; Sigma) stock solution at 10 mg/mL in saline was diluted 1000× in the culture medium and incubated for 60 min. After washing with 1 × PBS, cells were then fixed for 20 min in 4% paraformaldehyde (PFA) and permeabilized with 0.3% Triton X-100 for 10 min. After blocking with 10% goat serum in 1 × PBS for 1 h, cells were incubated with a primary rabbit antibody against BrdU (1:200, Abcam) over night at 4°C, and then incubated with the secondary antibody coupled to a fluorescent marker, Cy3, at room temperature for 2 h. After DAPI staining and 1 × PBS washing, the cover slips were mounted on to glass slides with anti-fade solution and visualized using a fluorescence microscope (Olympus 600 auto-biochemical analyzer, Tokyo, Japan) with Image-Pro Plus software for image analysis, and 10 microscopic fields were taken for calculating BrdU.
4
Quantifying Cell Proliferation via BrdU Assay
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A549 and SK-MES-1 cells were seeded on sterile cover glasses placed in the 6-well plates. After transfection with miR-206 mimic, miR mimic NC, miR-206 inhibitor, miR inhibitor NC for forty eight hours, the BrdU (5-bromo-2-deoxyuridine; Sigma) stock solution at 10 mg/mL in saline was diluted 1000 × in the culture medium and incubated for 60 min. After washing with PBS, cells were then fixed for 20 min in 4% paraformaldehyde (PFA) and permeabilized with 0.3% Triton X-100 for 10 min. After blocking with 10% goat serum in PBS for 1 h, cells were incubated with a primary rabbit antibody against BrdU (1:200, Abcam) over night at 4°C, and then incubated with the secondary antibody coupled to a fluorescent marker, Cy3, at room temperature for 2 h. After DAPI staining and PBS washing, the cover slips were mounted on to glass slides with anti-fade solution and visualized using a fluorescence microscope (Olympus 600 auto-biochemical analyzer, Tokyo, Japan) with Image-Pro Plus software for image analysis, and 10 microscopic fields were taken for calculating BrdU.
5
Cell Proliferation Analysis via BrdU Assay
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A549 and H1299 cells were seeded on sterile cover glasses placed in the 6-well plates. After transfection with miR-329 mimic, miR mimic NC, miR-329 inhibitor, miR inhibitor NC for forty eight hours, the BrdU (5-bromo-2-deoxyuridine; Sigma) stock solution at 10 mg/mL in saline was diluted 1000× in the culture medium and incubated for 60 min. After washing with PBS, cells were then fixed for 20 min in 4% paraformaldehyde (PFA) and permeabilized with 0.3% Triton X-100 for 10 min. After blocking with 10% goat serum in PBS for 1 h, cells were incubated with a primary rabbit antibody against BrdU (1:200, Abcam) over night at 4°C, and then incubated with the secondary antibody coupled to a fluorescent marker, Cy3, at room temperature for 2 h. After DAPI staining and PBS washing, the cover slips were mounted on to glass slides with anti-fade solution and visualized using a fluorescence microscope (Olympus 600 auto-biochemical analyzer, Tokyo, Japan) with Image-Pro Plus software for image analysis, and 10 microscopic fields were taken for calculating BrdU.
6
Cell Proliferation Assay Using BrdU
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HCT116 and SW480 cells were seeded on sterile cover glasses placed in the 6-well plates. After transfection with miR-875-5p mimic, miR mimic NC, miR-875-5p inhibitor, miR inhibitor NC for forty eight hours, the BrdU (5-bromo-2-deoxyuridine; Sigma) stock solution at 10 mg/mL in saline was diluted 1000× in the culture medium and incubated for 60 min. After washing with 1× PBS, cells were then fixed for 20 min in 4% paraformaldehyde (PFA) and permeabilized with 0.3% Triton X-100 for 10 min. After blocking with 10% goat serum in 1× PBS for 1 h, cells were incubated with a primary rabbit antibody against BrdU (1:200, Abcam) over night at 4°C, and then incubated with the secondary antibody coupled to a fluorescent marker, Cy3, at room temperature for 2 h. After DAPI staining and 1× PBS washing, the cover slips were mounted on to glass slides with anti-fade solution and visualized using a fluorescence microscope (Olympus 600 auto-biochemical analyzer, Tokyo, Japan) with Image-Pro Plus software for image analysis, and 10 microscopic fields were taken for calculating BrdU.
7
BrdU Labeling of Proliferating Cells
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A549 and SPC-A-1 cells were seeded on sterile cover glasses placed in the 6-well plates. After transfection with miR-134 mimic, miR mimic NC, miR-134 inhibitor, miR inhibitor NC for forty eight hours, the BrdU (5-bromo-2-deoxyuridine; Sigma) stock solution at 10 mg/mL in saline was diluted 1000× in the culture medium and incubated for 60 min. After washing with 1× PBS, cells were then fixed for 20 min in 4% paraformaldehyde (PFA) and permeabilized with 0.3% Triton X-100 for 10 min. After blocking with 10% goat serum in 1× PBS for 1 h, cells were incubated with a primary rabbit antibody against BrdU (1:200, Abcam) over night at 4°C, and then incubated with the secondary antibody coupled to a fluorescent marker, Cy3, at room temperature for 2 h. After DAPI staining and 1× PBS washing, the cover slips were mounted on to glass slides with anti-fade solution and visualized using a fluorescence microscope (Olympus 600 auto-biochemical analyzer, Tokyo, Japan) with Image-Pro Plus software for image analysis, and 10 microscopic fields were taken for calculating BrdU.
8
Evaluating Cell Proliferation and Inhibition
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Cell proliferation rate and inhibition rate were detected by MTT assay. CRC cells were harvested and seeded at a density of 1×10 3 cells/ml per well in 96-well plate and cultured at 37°C. After treatment, the cells were washed with PBS twice. Then, 10 μL MTT (5 mg/mL) was added into the wells at different time points. After incubation for 4 hrs, 100 μL DMSO was added to each well to dissolve the formazan crystal, and the absorbance was detected at 590 nm wavelength using a microplate reader (Bio-Tek, Winooski, VT, USA). In colony formation assay, 0.5×10 3 COLO205 and SW620 cells were inoculated into 6-well plates for 10 days. The colonies were fixed with 10% formaldehyde for 10 min and dyed with 0.5% crystal violet for 5 min. The number of colonies was photographed by an Olympus microscope (Tokyo, Japan) and counted.
BrdU immunofluorescence assay CRC cells were seeded on coverslips placed in 6-well plates and incubated with BrdU (Sigma) stock solution for 60 min. After blocking with 10% goat serum in PBS for 1 h, cells were incubated with a primary rabbit antibody against BrdU (1:200, Abcam) overnight at 4°C, and then incubated with the secondary antibody coupled to Cy3 for 2 hrs. After DAPI staining and PBS washing, the coverslips were visualized using a fluorescence microscope (Olympus 600, Tokyo, Japan) and BrdU-positive cells were counted in five fields of each coverslip.
BrdU immunofluorescence assay CRC cells were seeded on coverslips placed in 6-well plates and incubated with BrdU (Sigma) stock solution for 60 min. After blocking with 10% goat serum in PBS for 1 h, cells were incubated with a primary rabbit antibody against BrdU (1:200, Abcam) overnight at 4°C, and then incubated with the secondary antibody coupled to Cy3 for 2 hrs. After DAPI staining and PBS washing, the coverslips were visualized using a fluorescence microscope (Olympus 600, Tokyo, Japan) and BrdU-positive cells were counted in five fields of each coverslip.
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