During FISH, we have consistently had issues getting signals FITC (flourescein) probes while Cy3 and Cy5 signals have been much easier. The weak or non-existent FITC signal is observed independent of what organism is being targeted or the probe design, so I suspect it has something to do with the methodology when using FITC probes. Has anyone else had issues with FITC probe signals? What have you done to resolve the issue?
In doing my own research I found one recommendation: https://www.arb-silva.de/fish-probes/troubleshooting/
"for probes labeled with fluorescein derivatives, check if pH of mountant/resuspension buffer exceeds pH 9.0 (fluorescence maximum of fluorescein is pH >9.0)" I also noted that thermo-fisher suggests that flourescein is very sensitive to pH. So maybe my problem is related to the pH the probes are exposed to?
After we recieve the commercially synthesized probe, I re-suspend the dry probes in DNAse/RNAse free water and I use TE pH = 8.0 for the hybridization/wash buffers. What has worked for your labs?
Hi Kathrin,
I'm usually using the more robust Atto dyes lately. They're much more hassle-free I find. Here have a look:
https://www.sigmaaldrich.com/technical-documents/articles/biofiles/atto-dyes-for-superior-fluorescent-imaging.html
I have used HuluFISH probes for my experiments where they directly synthesize the probes of choice with a respective Atto dye. I was happy with the results so I even freelance for them now. Let me know if you're interested.
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