Hello, I am desperately in a need for advice on RNA in situ hybridization.
I tried two protocols from Fire and from Kohara labs. Both did not work well for me. I am getting a lot of background staining that is non-specific and very little specific staining.
I am using DIG labeled probes and detect them with alkaline-phosphatase anti-DIG antibody or fluorescein conjugated anti-DIG antibody.
I troubleshoot a lot at different steps, but still with no success.
Any help would be greatly appreciated.
I can provide images from unsuccessful rounds to anyone willing to help.
Thanx
daniel
Dear Daniel,
We have developed a protocol that seems to work very well for embryos (and it could be adapted for larvae). It is our plan to make it available very soon through the WormBook, but if you would like the protocol sooner, please contact me.
Regards,
Morris
mmaduro@ucr.edu
I was just about to start the same procedure. Does any one know, what is best probe to use? RNA? DNA single stranded? Oligo?