I recently started working on ISH (non radioactive on PEFF tissues) for Retinoblastoma RNA in Dog osteosarcoma.We have created the probes from the mRNA bank in gene Bank. We created the plasmid (TOPO4) and after sequencing there were very few mismatch point and the homology was very high also with other species (98% in the cat). Anyway the probe is about 900 bases long. By dot blot the polimerization appeared to have come out without problems. Anyway I have not obtained any specific staining yet. In the lab were I am courrently working on my in situ they told me that for infectious disease also 850 and slightly above probe reacted very well. Now is it true that the infected cells contain usually an higher quantity of infectious’s agent mRNA, and that the cellular mRNA usually is present in lower doses, but I’d like to know if other people here who worked with ISH in tumors could help me to better asses my protocol. I tried to amplify the signal with biotinilated anti digoxigenin antibodies, to which streptavidine alcaline phospahtase was added. But no signal was present though. Again I tried to linearize the RNA by incubating for 10 minutes at 70 degree the slides with the prehybridization solution and the hybridization solution at 90 for 5 minutes follwed by a chill bath in ICE. But not results so far. Incubation T is 42C overnight. I used up till 200 microgram/ml, but so far there was any canges betwen the 100 that I sd in other experiments.
I’d like to know if for you it is mainly a problem of probe lenght, or maybe I should change other protocol’s variables, and see what happens.
We ordered new primers to do a shorter probe. But in the meantime I’d like to work on other variables in the protocols. Again the tisues I runt the most of my experiments on were not decalcified and it showed a very string positivity to pRB by IHC.