We use FISH with commercial probe and hybridisation buffer and with mouse bone marrow cells. We have interphase and metaphase nuclei as well. We use 72 C for 2 min for co-denaturation, then 24 h hybridization in 37 C, then wash in 72 C for 2 min (0,4x SSC, 0,3% NP-40), then in room temperature for 1 min (2x SSC, 0,1% NP-40). We have tried with separate denaturation, but the result was the same. What could cause the lack of specific signals? Thank you.
I would try to prolong the denaturation step with at least 10 min. at 75°C.
Hi Rita,
Have you tried denaturing the probe separately?
Your nuclei looks overdenatured. I experience overdenaturation when I denature my cells at 70-72 degree C for 2 minutes but the best results I have obtained by far is denaturing my cells at 72 degree C for around 1 minute 50 seconds. Your probes are DNA which can withstand a much higher temperature. It may be that your probes cannot bind because they are not well-denatured.
As Torsten suggested, you could co-denature at a higher temperature around 75-76 degree C and it would help, however, you would also have very poor quality nuclei.
The alternative is to do the separate denaturation steps then hybridize. Your commercial probe should have separate instructions for the alternative procedure for separate denaturations. It is likely the probe would be denatured at around 85-90 degree C, then you chuck it on ice before adding it to your slides.
The reason your probe may need a higher temperature for denaturation is the construct itself. Since GC-rich regions will need a higher melting temperature, the normal calculations would be 2 degree C for A-T and 4 degree C for G-C. Depending on the combination of ATGC in your probe itself, the melting temperature would differ.
Hope that helps. ^^
if u are using vysis probes and a hybrite 72 degrees and 2 minutes for denaturation should give u good signals....but if u are using any other probe as cytocell or kreatech and a hybride as leica use 75 degrees and 5 minutes for denaturation it will give u good signals...this is what works in my lab
Dear Rita
In my opinion with Vysis the time of codesnaturalization is ok. We do the second wash at room temperature but only for 10 seconds, we have good results in metaphases and interface.
luis
hi
after the second wash we wash the slides in water a few seconds.
luis
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