in situ hybridization question - (May/09/2012 )

Hi all,

I'm working with ISH on zebrafish embryo right now (DIG-lebeling). In the end I will need to develop multiple probes ISH (actually 3 probes altogether) which means I will need all the probes to have approximately same color development period as well as annealing temperature. When I did ISH of each probe separately on the embryo, 2 of them works fine, but the third one need a significant longer color development period and that generated a high background.

I was wondering how can I decrease this background and shorten the color development period of this probe? It's pax2, by the way.

Thank you very much for all your great help and suggestion. Any further information needed, please tell me.

Daisy

I'm actually starting ISH myself, unfortunately in adult sectioned tissue which is obscenely difficult for no apparent reason, but question, are you using directly conjugated anti-dig fragments from roche?
If you are not using directly conjugated antibodies, some form of signal amplification before the color reaction should help with both reaction time and background. For a low level transcription factor like pax2, you might need something like TSA amplification prior to color reaction.

Also I'm a bit confused, are you doing triple color ISH simultaneously using dig, fitc, and biotin?

blin100 on Sat May 12 20:29:26 2012 said:

Ah, I see now what the problem is.
I can't really see any way of specific signal amplification since you are using a single detection method, and increasing probe concentration for one will simply increase background, and any steps you use to remove background for that one probe will affect all the others.

Just thinking off the top of my head, you could use 2 different detection methods, such as HRP-conjugated anti-Dig and AP-anti-Dig. Use one to detect two of the substrates, and the other to detect the one thats being troublesome.

I'm not sure of the feasibility of fluorescent ISH in zebrafish embryo, but that would be the other way, where you make your probes with fitc, dig, and biotin tagged nucleotides. Instead of color development, you then use, for example, Donkey-anti-fitc, goat-anti-dig, and SA antibodies. The final step is to use the appropriate conjugated antibodies for detection, for example fitc-anti-Donkey, red-anti-goat, and blue-biotin/sa. (If you really didn't care about the color, this last step could just be antibodies of the same color.)

The main reason for any of this would be to gain the ability to modify amplification of your Pax6 probe, and not worry about screwing the other stuff up.
Unfortunately, after typing out all of this, this is pretty involved if your lab is not setup for this already, I'm not sure how important this triple staining is

Perhaps if you simply made two of your probes dig, the pax one biotin labeled. Do everything as now, but do TSA amplification for the pax probe. Then color develop all three using AP or whatever you are using now?