I'm doing FISH analysis in fecal samples using different probes labelled with either Cy3 or FITC or DAPI. I'll use different filter cubes to detect each probe in the sample, and finally overlay the images. My question is... Can I use all probes with different labels in the same hybridization step?
Theoretically yes. Doublestaining in FISH is done regularly. Practically you have to work with probes, that need the same conditions in denaturation, hybridization and stringent washing. Otherwise one probe could not bind, while another is washed away.
I only worked with commercial products like Her2/Cep17 plus nuclei-staining with Dapi. To develope an own FISH protocol for multistaining may need some trial and error.
Yes ,Thaisa. I have made cytogenetic diagnosis in chromosomes using probes from 21,13, 18, X and Y . All this in one hybridization step. You should careful with the mixture of probes and the fluorocrome(color) of each probe.
luis
The formamide concentration is the same. However, one of the probes requires a pre treatment with lyzozyme before hybridization. Do you think it may interfere in the hybridization of the ones that doesn't need this pre-treatment?
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