How to dissolve DAPI?

Hello Aigerim Kabulova

You may prepare the stock solution of DAPI (5 mg/mL) in dH2O. However, subsequent dilutions can be made with water or McIlvain’s buffer pH 4. I have doubts using PBS to make further dilutions.

You may prepare McIlvain’s buffer pH 4 as provided in the link below.

https://www.wikiwand.com/en/McIlvaine_buffer

Best.

Ahmad Al Khraisat

@all If you are observing small aggregates and poor staining intensity after diluting your DAPI stock solution, there are a few potential issues you can investigate and steps you can take to improve your staining results:

Aggregation: Aggregation of DAPI can occur when the concentration is high or when there are impurities in the solution. Try centrifuging the diluted solution at a low speed (e.g., 500-1000 rpm) for a short time (e.g., 1-2 minutes) to remove any aggregates. This step should help improve the staining quality.

Filtering: It's possible that your stock solution or diluted solution contains particles or impurities that are contributing to the aggregation and poor staining. Consider filtering your stock solution and/or the diluted solution using a syringe filter with a pore size appropriate for removing particulates (e.g., 0.2 μm or smaller). This can help remove any unwanted particles and improve the staining quality.

Storage conditions: Check the storage conditions of your DAPI stock solution. DAPI is sensitive to light and can degrade over time if exposed to prolonged light exposure. Ensure that your stock solution is stored in a dark container, and protect it from exposure to light during handling and storage.

Dilution ratio: The dilution ratio can also affect the staining intensity. If the staining intensity is too low, you may consider adjusting the dilution ratio to increase the concentration of DAPI in the staining solution. You can try diluting the stock solution to a higher concentration, such as 500 nM or 1 μM, and then adjust accordingly to find the optimal staining intensity.

Time and temperature: Optimizing the staining protocol can also improve the results. Ensure that you are incubating the fixed cells with the diluted DAPI solution for an adequate amount of time and at the appropriate temperature. Typically, incubating the cells with the staining solution for 5-15 minutes at room temperature or 37°C is sufficient, but you may need to optimize the timing based on your specific experimental conditions.

By following these suggestions, you should be able to improve the staining quality and reduce the aggregation of DAPI in your solution.

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