Hi, I am doing FISH on cancer cells, my problem is that control signals were visible but not in test signals though I know that test samples are strongly positive. My problem is that if the test samples are positive then what could be possible reason behind it. If controls showing signals then test signals should also come. please help me out with your ideas.
Could you have mixed the control and test samples up by accident? Also, when you say you know the test samples are strongly positive, is this because you've seen them be positive before or because the literature says they should be? If it's literature-based, are you using the same protocol they did? If not, this could also be why there is a problem.
I don't mean my answers to sound patronizing, but sometimes it's the small things we don't think about that are the problem.
Let me know a bit more about how you're doing the FISH and perhaps we can come to a resolution!
The working of the FISH method is strongly dependant on the fixation of the specimen. How do you fix the cells? I made my experience with formalin-fixed tissue. We stained several bits of tissue on one slide and saw different outcomes from bit to bit. Make sure, that your positive-control has the same pretreatment (fixation, processing) as your specimen.
Besides the fixation, the pretreatment-conditions played a big role. The temperature varied from the bottom of the slide to the upper end (vertical slide in the waterbath). So at the bottom we had strong signals and on the upper end we had hardly.
Repeat the test with longer digestion until the cells look really ugly. ; )
Thankyou so much Lang,
Is it possible that the siignals of CEP17 were comng but not for HER2 signals in the same slide?
Yes. The centromer probes bind to a longer area and signals are stronger. Try to prolong the digestion. If nuclei look still very dense, a too short digestion may be the cause.
Keep in mind also that the sequence of the probe can also contribute to its ability to detect the target sequence in the cell. Keep a close eye on the Tm of the test probes and your control probes as this can also vary and cause differences in detection.
for this issue, first you need to ensure that the cell preservation was identical. Most of the universal weak signal problems are related to cell preservation. If you treat the tissue side by side, the procedure should be the same. you may also try to optimize the digestion, denature temperature, and incubation time. Good luck.
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