Hi
I've been doing whole mount ISH on E9.5-10.5 mouse embryos (with DIG-labeled RNA probes). For a long time, I've been struggling with getting a decent signal before the background starts to become too strong (NBT/BCIP). On gel electrophoresis, the RNA probe seems ok, so I'm assuming the probe quality is ok and I've included several steps to reduce background (levamisole, acetic anhydride in TEA, RNAse).
After sectioning some embryos that were hybridized for a cardiac specific probe and showed a signal in the heart as well as some overall background, I found some weak expression in the heart, but also a very strong signal all over the embryo surface.
Has anyone seen this before? I've googled google extensively, but I have not been able to find a convincing answer. I suspect it may be residual background signal, but am also considering overfixation/suboptimal protK treatment causing suboptimal penetration of the probe (even though I do a 9-10 minute incubation with 10 ug/ml prot K at room temperature).
Thanks.
Thanks.
The probes I have are anywhere between 700-1000+ bp. As I understood it, this is supposed to increase specificity, but penetration may indeed have been a bigger issue than I anticipated.
The washing is something I've noticed. I do most of my washes in 5 ml glass vials now and spread it out over two days (between antibody and coloring).
I normally don't puncture anything until I have rehydrated the embryos again, assuming that fixation would be good enough without puncturing the embryos (it is sufficient for my standard histology, so I assumed the fixative doesn't have much of an issue with penetration).
In any case, if I do get a somewhat specific signal, it remains weak and takes several hours to develop (at RT), so apart from reducing background, I obviously need to increase the signal to improve the signal/noise ratio.
Best,
Piet
Thank you. The cardiac probe I'm testing is Nkx2.5. It was supposed to work as a control actually for the entire procedure and from what I can find in other publications, it should be very cardiac specific. The probe itself is only 700 bp, so not extremely long.
I'll try the increased protK, extensive washing, and BM purple. NBT/BCIP coloring and background also seems to vary with the NBT/BCIP ratio, but most protocols appear to simply follow the manufacturer's instructions without much suggestions for how to improve the signal/noise ratio.
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