The protocol for in situ that I use has been giving me good signals without too much non-specific background until now. This time using a different set of same probes while doing exactly the same washes gave me an overwhelmingly high red background signals. I used centromere and a high copy locus specific probe with biotin label and detected using texas red conjugated antibodies cascade.
I tried to look up information for troubleshooting, and found some reasons that might cause high non-specific signals, which in my case seems unlikely. Besides drying out of slides, degradation of formamide solution and large sized probe DNA fragments, what other reasons causes an overwhelmingly high non-specific signals in FISH (in my case biotin/texas red signals)? I have also attached an image to see how it looks when all the channels are merged.
1. Based on your picture i could make out patched debris on the spread. You can try to air dry the slides as soon as possible, which means after you removed the slides from alcohol series, blot excess alcohol with tissue and wave the slide fast, so the minute particles which stick on the spread will move out and then when washing it will go completely.
2. If possible try to reduce the probe amount and check
Dear Meenakshi,
Thank you for your valuable suggestion! I found out what my problem was. My centromere probe is a repeat, which is PCR labeled, so when I PCR labeled it also had a smear of large sized fragment (extending much higher than 10 Kb) and because of this large fragments, my probe was sticky everywhere and was giving me background signals.
Sirjan
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