How can I improve DNA FISH signal consistency and reduce signal loss?

19 June 2025 0 2K Report

Hi everyone,

I’m developing a DNA FISH protocol to detect an expanded repeat region in a mammalian cell line. The repeat tract is known to be long (several hundred copies), and the cells are expected to carry around 800 copies of the repeat.

I'm using a short probe (4-repeat unit) labeled with AF488, and co-denaturation of probe and genomic DNA at 95 °C for 30 minutes.

However, I’ve encountered a few issues:

Hybridization buffer issue: Initially, I accidentally used only 1% dextran sulfate in the hybridization buffer. After correcting it to 10%, I expected improved signal, but surprisingly, the number of positive cells dropped. Could this change affect probe accessibility or hybridization kinetics?

Reproducibility problem: The method is unstable, signal quality and intensity vary greatly between experiments, making quantification difficult.

Signal amplification: Are there recommended signal amplification methods that work well for repetitive DNA FISH, especially for GC-rich regions?

Any suggestions or insights would be greatly appreciated. Thank you very much!

Best,

Raven

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