5-BrdU

This protocol only provides a guideline, and should be modified according to your specific needs.
1. Solution preparation
 
(1) Stock solution: Dissolve 5-BrdU solid in DMSO or deionized water to a final concentration of 10 mM and filter sterilize.
Note: After unused storage solution is aliquoted, store it in a dark place at -20℃ to avoid repeated freezing and thawing.
 
(2) Working solution: Before the formal experiment, dilute the stock solution with experimental buffer to the required working concentration and filter sterilize.
Note: Please adjust the working concentration of a specific application according to the actual situation or refer to the literature to set the gradient concentration by yourself to explore the best conditions. The working solution must be prepared and used immediately.
 
2. 5-BrdU in vitro labeling of cells
 
(1) Weigh 3 mg 5-BrdU and dissolve it in 1 ml deionized water until it is fully dissolved. Prepare 10 mM BrdU stock solution. Dilute the stock solution to 10 μM BrdU staining solution with cell culture medium at a ratio of 1:1000, and filter sterilize the staining solution with a 0.2 μm filter under sterile conditions.
(2) Aspirate the intracellular culture medium and replace with sterile 10µM BrdU staining solution, and incubate in a CO₂ incubator at 37℃ for 1-24h.
(3) Aspirate the intracellular staining solution and wash twice with PBS, each time for at least 5s.
(4) Fix and permeabilize the cells according to the standard immunocytochemistry (ICC) operation process. However, before starting immunostaining, refer to the following DNA denaturation steps.
 
Note: The 5-BrdU incubation time depends on the cell division rate. Primary cells may take up to 24h, but fast-proliferating cell lines may only take 1h. The time to achieve the best signal-to-noise ratio needs to be determined by optimizing conditions.
 
3. 5-BrdU in vivo labeling
 
There are many methods for 5-BrdU in vivo labeling, and the most common one is intraperitoneal injection. The following are the steps for intraperitoneal injection. Other methods can refer to the literature or operate according to the inherent experience of the laboratory.
(1) Dissolve an appropriate amount of 5-BrdU in 1x PBS to prepare a 10 mg/ml stock solution, filter and sterilize, and freeze it in single-use aliquots for later use. Avoid repeated freezing and thawing.
(2) For mice, the commonly used injection concentration of 5-BrdU is 100 mg/kg. After labeling, treat the animals according to established laboratory procedures.
 
Note: 5-BrdU insertion into rapidly differentiating tissues such as the small intestine can be detected 30 minutes after injection. However, most tissues may require up to 24 hours to be detected. The appropriate treatment time and injection dose need to be optimized based on the tissue type.
 
4. DNA denaturation step
Cell sample:
(1) Incubate cells with 1-2.5M HCl at room temperature for 10 minutes to 1 hour. The actual HCl concentration and incubation time are optimized based on the experiment. If a shorter incubation time is used, incubation at 37°C may be more effective than incubation at room temperature.
(2) Optional step: Remove HCl and neutralize with 0.1M sodium borate buffer, pH 8.5 at room temperature for 30 minutes.
(3) Wash with PBS three times, each time for at least 5 seconds. Continue with immunostaining according to the standard IHC procedure.
Tissue sections:
(1) Dewax paraffin sections first.
(2) Incubate sections with 1-2.5M HCl at room temperature for 30 minutes to 1 hour. The actual HCl concentration and incubation time are optimized according to the experiment. If a shorter incubation time is used, incubation at 37°C may be more effective than incubation at room temperature.
(3) Optional step: Remove HCl and neutralize sections with 0.1M sodium borate buffer, pH 8.5 at room temperature for 10 minutes.
(4) Wash with PBS three times, each time for at least 5 seconds. Continue with immunostaining according to the standard IHC procedure.
 
Note: 5-BrdU is a mutagen. Be sure to take precautions during the operation and avoid direct contact with the skin. In addition, wear a mask to avoid inhalation of dust. For your safety and health, please wear a lab coat and disposable gloves.