I am doing In situ hybridizations on zebrafish cryosections (embeded in OCT) and I can't make them work. I use to follow the same protocol one year ago and it worked perfecly, but now I can't see signal.
I tried everything that has come up to my mind: make all the solutions new, synthesize new probes, try other samples, increase the signal developing time...
Last in situ try was the most confusing, since the probe that I used as a control probe worked but the probe that I was interested in didn't show up, not even a shadow. Normally I do an overnight to let the signal develop. For the control probe this was enough, but for my probe it wasn't so I left the development even overweekend at 4ºC. No signal apeared.
I am really desperated, since the ISH for the gene I am studying used to work and now it doesn't.
Any suggestions of what is happening?
Did you check that your probe is not degraded? I usually run few microlitres on a fresh gel to see that the probe is ok.
I know that you synthesize new probes, so they should be fine but maybe some of your reagents are contaminated and the probes get degraded.
But the control probe did work, so the reagents of the ISH protocol are not contaminated. ONe of the probes tested against my gene did work in an embryo WISH experiment
Yes, but if only the probe of your gene is degraded not because of ISH reagents, the control will still give you results. But I guess that if it worked in another experiment, this is not the case.
You mentionned you tried synthesizing new probes. Are you certain the synthesis worked? We recently ran into trouble with our probe synthesis. Our issue was that a component in our PCR kit (that we used to first amplify our sequence of interest) inhibited the subsequent reverse transcription.
Do you treat your samples with methanol after fixation and before cryosectioning?
After the probe synthesis I always run a dot blot, and all the probes were properly labelled.
I always treat the samples with methanol (dehydrating and rehydrating) after fixation and before cryosectioning.
Was the sequence of the probe that you are interested designed by you or from other lab? If designed by you, did you try to use different part of the gene such as the 3' UTR, shorter or longer of your probe sequence? I've been the similar situation like you, I shortened my probe and increased the consentration, from that I got stronger signals. And for sectioned insitu, I'd like to do color development at RT if no background appears, at 4 degree sometimes let the color development last for ever. If the probe from other lab, you'd ask suggestion from them about tricks of this perticular probe. Good luck!
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