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BrdU detection kit for DNA Detection 100T Cell proliferation detection reagent

Volume: 100T

 

  • G4102-100T
  • Servicebio

Product Description

Product Information

 

Product Name

Cat. No.

Specification

BrdU detection kit

G4102-50T

50T

G4102-100T

100T

 

Product introduction:

 

This product BrdU detection kit is used for cell proliferation detection of cells and tissue sections. BrdU (5-Bromo-2-deoxyUridine), which is 5-bromodeoxyuridine, is an analog of thymidine (T). The principle of this kit is that BrdU can replace thymidine T by competition. Enter the DNA synthesis phase (S phase). When BrdU is incorporated into cells in vigorous division, the cells containing BrdU can be detected by using anti-BrdU antibody and antibody ligase or fluorescein as an indicator system, and combined with other cell markers for dual labeling, the proliferation of cells can be judged Types, proliferation speed, etc. are of great significance to the study of cell dynamics. BrdU enters tissue cells through intravital injection or cell culture, and the DNA incorporated into it is accurately positioned, which can effectively reflect the level of newly synthesized DNA in S-phase cells, and is less affected by the internal and external environment of the cell, and the label will not be lost.

 

Storage and transportation

 

Ice bag (wet ice) transportation; rupture fluid, blocking fluid (3% BSA), 1 M HCl can be stored at 4°C, 4% paraformaldehyde can be stored at room temperature, anti-BrdU (mouse mAb) needs to be stored at -20°C, expiration date 12 months.

 

Component

 

Component Number

Component

G4102-50T

G4102-100T

G4102-1

Rupture fluid

30 mL

30 mL

G4102-2

Sealing fluid (3%BSA)

30 mL

30 mL

G4102-3

4% Paraformaldehyde

30 mL

30 mL

G4102-4

1 M HCl

30 mL

30 mL

G4102-5

Anti-BrdU (mouse mAb)

50 μL

100 μL

Product User Manual

1pc

1pc

 

 

Experiment preparation

 

1. Bring your own PBS buffer (recommended G4202), secondary antibody, gradient ethanol, mounting tablets, etc.;

2. Bring your own nuclear staining solution, recommend G1004 hematoxylin stain or G1012 DAPI stain (ready-to-use);

3. Prepare anti-BrdU primary antibody working solution: Dilute anti-BrdU (mouse mAb) with blocking solution (3% BSA) 100 times to prepare anti-BrdU primary antibody working solution, prepare it for current use, store at 4℃;

4. Secondary antibody working solution: Prepare your own HRP-labeled (subsequent white light detection, DAB color kit, recommended G1212) or fluorescently labeled (subsequent fluorescence detection) anti-mouse secondary antibody, and dilute with PBS to prepare Secondary antibody working fluid.

 

Operation Steps:

Cell sample:

 

1. Cell preparation: prepare cell slides in advance to ensure that the cells are in normal condition and adhere well;

 

2. BrdU incubation: Cells are incubated with BrdU-containing medium in an incubator in the dark for a certain period of time. The concentration of BrdU and the incubation time of cells are different depending on the cell type. It is recommended to explore the best conditions in pre-experiment. The experimental conditions of the tested cells in the precautions are for reference;

 

3. Cell fixation: The above-mentioned cells were incubated with BrdU and washed 3 times with PBS for 5 minutes each time. Add 1 mL of 4% paraformaldehyde to the orifice to cover the cells, and fix for 20-30 min at room temperature. Wash with PBS 3 times, 5 min each time;

 

4. Permeability: Add rupture fluid to the orifice to cover the cells for 8-10 minutes, wash with PBS 3 times, 5 minutes each time;

 

5. Nuclear staining (optional):

 

For subsequent white light detection, stain the cell nucleus with hematoxylin: add hematoxylin staining solution to the well plate for 5 min at room temperature, aspirate the hematoxylin staining solution, and wash 3 times with PBS for 5 min each;

 

For subsequent fluorescence detection, stain the cell nucleus with DAPI: add DAPI dye solution to the well plate for 5 min at room temperature, aspirate the DAPI dye solution, and wash 3 times with PBS for 5 min each time;

 

6. Re-fixation: Add 4% paraformaldehyde dropwise to the well plate and incubate for 10 min at room temperature, wash 3 times with PBS, 5 min each time;

 

7. Acidification: Add 1 M HCl dropwise to the well plate to cover the cells, treat at 37°C for 10 min, wash 3 times with PBS, 5 min each time;

 

8. Post-fixation: add 4% paraformaldehyde dropwise to the well plate again, incubate at room temperature for 10 min, wash with PBS 3 times, 5 min each time;

 

9. Blocking: Add blocking solution (3% BSA) dropwise to the well plate and incubate for 30 min at room temperature. Absorb and discard the blocking solution, do not clean;

 

10. Anti-BrdU antibody incubation: Add anti-BrdU primary antibody working solution dropwise to the well plate to cover the cells, and incubate overnight at 4°C. Aspirate and discard the anti-BrdU primary antibody working solution, wash with PBS 3 times, 5 min each time;

 

11. Secondary antibody incubation: Drop the secondary antibody working solution into the well plate to cover the cells, and incubate at room temperature for 1 h. Aspirate and discard the working solution of the secondary antibody, and wash with PBS 3 times, 5 min each time. Note that if you use a fluorescently labeled secondary antibody, you need to avoid light during incubation and washing;

 

12. Covering and microscopic examination:

 

If it is an HRP-labeled secondary antibody, use DAB chromogenic reagent (G1212 recommended) to develop the color of the cell sample, dehydration, and transparency. Use a pointed tweezers to gently pick up the slide and mount it upside down on a clean glass slide. , Microscope observation;

 

If it is a fluorescently labeled secondary antibody, drop an anti-fluorescence quenching mount (G1401 is recommended) on the clean glass slide, gently pick up the slide with pointed tweezers, buckle upside down on the glass slide and mount the slide, observe with a fluorescence microscope ;

 

Tissue section samples:

 

1. Animal preparation: prepare experimental animals in advance and carry out BrdU labeling in vivo, please refer to WGB8010. After labeling in vivo, take materials, fix and prepare paraffin sections according to experimental needs;

 

2. Tissue paraffin sections are deparaffinized and rehydrated;

 

3. Repair: Group the brush to circle the tissue, immerse the slice in the antigen retrieval solution, and repair by microwave heating. Natural cooling

 

4. Nuclear staining (optional):

 

For subsequent white light detection, stain the cell nucleus with hematoxylin: add hematoxylin staining solution to the well plate for 5 min at room temperature, aspirate the hematoxylin staining solution, and wash 3 times with PBS for 5 min each;

 

For subsequent fluorescence detection, stain the cell nucleus with DAPI: add DAPI dye solution to the well plate for 5 min at room temperature, aspirate the DAPI dye solution, and wash 3 times with PBS for 5 min each time;

 

5. Re-fixation: Add 4% paraformaldehyde dropwise to the tissue and incubate for 10 min at room temperature, wash 3 times with PBS, 5 min each time;

 

6. Acidification: Add 1 M HCl to the slices to cover the tissues, treat them at 37°C for 10 min, wash them with PBS 3 times, 5 min each time;

 

7. Post-fixation: add 4% paraformaldehyde dropwise to the well plate again and incubate for 10 min, wash with PBS 3 times, 5 min each time;

 

8. Endogenous peroxidase quenching (optional): Add 3% H2O2 dropwise to the slices and incubate at room temperature for 25-30 minutes to remove endogenous peroxidase. Then the slices were washed 3 times with PBS, 5 min each time;

 

9. Blocking: Add blocking solution (3% BSA) to the slices to cover the tissue, and incubate at room temperature for 30 minutes. Remove the blocking fluid, do not clean;

 

10. Anti-BrdU antibody incubation: Add anti-BrdU primary antibody working solution to the slices to cover the tissue, and incubate overnight at 4°C. Remove the anti-BrdU primary antibody working solution, wash 3 times with PBS, 5 min each time;

 

11. Secondary antibody incubation: Add the working solution of the secondary antibody to the section to cover the tissue, and incubate at room temperature for 1 h. Remove the working solution of the secondary antibody, wash 3 times with PBS, 5 min each time. Note that if you use a fluorescently labeled secondary antibody, you need to avoid light during incubation and washing;

 

12. Covering and microscopic examination:

 

If it is HRP-labeled secondary antibody, use DAB chromogenic reagent (G1212 recommended) to develop the color of the tissue section, dehydrate, transparent, and observe under microscope after mounting;

 

If it is a fluorescently labeled secondary antibody, add dropwise anti-fluorescence quenching mounting tablets (G1401 recommended) to mount and observe with a fluorescence microscope.

 

Observation of Results

 

If detected with HRP-labeled secondary antibody, the nucleus is dark blue, and the proliferating cells are brown. If it is a fluorescently labeled secondary antibody, the nucleus will show blue fluorescence (Ex=358 nm, Em=461 nm), and the proliferating cells will show the fluorescence of the corresponding secondary antibody.

 

Notes:

 

1. For cell slide samples, the concentration and processing time of BrdU are related to the cell types. Generally speaking, the concentration and time required to treat tumor cells are low. Cells with slow proliferation such as fibroblasts or epithelial cells need to increase the concentration and prolong the action time. The recommended concentration range is 20-100 μM and the action time is 40 min. -4 hours, can be adjusted according to the specific cell types.

 

The following table shows the commonly used cell treatment concentration and time tested, for reference only.

 

Cell

BrdU concentration (μM)

Incubation time (min)

A549

40

40

Hela

40

40

NRK-52e

40

40

SKOV3

80

120

H9C2

40

40

 

2. In order to ensure the best experimental results, it is recommended to use other supporting reagents produced by our company: hematoxylin stain (G1004); DAPI stain (G1012); DAB color reagent kit (G1212), anti-fluorescence quenching mounting tablets ( G1401);

 

3. For your safety and health, please wear lab coats and disposable gloves for operation.

 

The product is for scientific research purposes only, not for clinical diagnosis!

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