I am performing a whole mount in situ hybridization on zebrafish embryos. I have increased hybridization T to 65C but am still getting nonspecific binding. Can I increase the T even higher and what would be the max hybridization T someone would use on zebrafish embryos?
Also, what times do you use for permabilisation?
Thank you
Hi Monika,
I've never done ISH on zebrafish but if you have non-specific signal then you probably will be better with increasing the pre-hyb duration.
Also, a more stringent wash post-hyb with the high-salt and low-salt SSC would improve your signal while getting rid of the non-specific binding.
Additionally, you might want to check if your probe has significant similarity with any other gene or isoform. Many genes also have a non-coding RNA being transcribed from the same locus and this could also explain if you notice signals elsewhere.
Goodluck with your experiments!
Navneet
Hi Monika! By personal experience, I know that the hybridization T can reach 70 C. Good luck with your experiments! Natascia
Hi,
I have used Tm till 70, it also depends on the annealing temperature of your primers. Also check the sequence of your oligoes if they have any off targets. You may increase stringent washes. Also try increasing the prehybridisation time. Though I have used Rat tissue, so the permeabilisation time considerably depends on the thickness of your sample.
All the best!
I always do my ISH on ZF at 70C but depends a bit on the size of your probe.
Good luck
I work with zebra finch tissue, but I found that doing an RNase treatment after the post-hyb washes dramatically reduced the amount of nonspecific labeling.
Following overnight hybridization I washed my tissue 2x5min in 2X SSC at 60 degrees C. Then I incubated the slides in 20μg/ml RNase in 2X SSC at 37 degrees C for 30 min. This breaks down any unbound RNA and for my work resulted in clean labeling when previously I had seen high background and nonspecific labeling. I followed this with 15 min rinse in 0.2X SSC at 37, then 3x5min in 0.2X SSC at 60, then continued on with the rest of the protocol as normal. If there is more detail that would be helpful I'm happy to share.
Hi Monika
I guess you have it figured out by now. But just in case let me provide you with some additional info -in case you don't have it already! :-)
There are some formulas to calculate your Tm, for example:
DNA-RNA hybrid (a,b,) Tm= 79.8°C + 18.5(logM) + 0.58(%GC)
+ 11.8(%GC)2 -0.50(%form) -820/L
a. M: molarity of monovalent cations (Na+ concentration); % GC: % G and C nucleotides in the DNA; % form: percentage of formamide in the hybridization solution; L: length of the duplexin base pairs; N: chain length.
b. These equations hold for Na+ concentrations between 0.01 and 0.40 M and % GC values of 30-75%.
(please check the website below)
And then, we try incubating with the probe 25ºC below Tm and do the washes 12ºC below Tm
I have included a few links for reference, just in case.
I hope any of this is useful!! ;-)
Best of luck!
http://dx.doi.org/10.1016/S0091-679X(08)60630-5
http://www.genomics.agilent.com/files/Mobio/Nucleic%20Acids_Equations_for_Calculating_Tm_3pgs.pdf
http://elsolbogota.co/free/in-situ-hybridization-protocols/
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