Can some recommend a protocol for ISH on paraffin sections?
I have tried two different protocols so far to no avail.
I am using the DIG RNA Labeling Kit (SP6/T7) from Roche to synthesize my probe. I don't think the problem lies in this step. I saved all steps of the synthesis to run on a gel and it looks fine. The probe also has worked on embryonic whole mount ISH. The probe is ~1.4 kb
I have tried this protocol from abcam. http://www.abcam.com...g...e&rid=11743 Only changes made were to add Levamisole to the MABT. Tried different ProK concentrations and treatment times.
I have also tried to adapt the whole mount protocol we have successfully used to parrafin sections. The whole mount protocol is slightly different. The hyb buffer contains SSC, CHAPS, Tween20, yeast tRNA, and EDTA instead of the salts sol'n, denharts, dextran sulfate, and SDS in abcams protocol. The protocols are similar. Whole mount one has a 4% PF fixation step instead of acetic acid. The post hyb washes are somewhat different but serve the same purpose. Development is with BM purple on both.
Lastly, I have de-paraffinized a few slides and scraped the tissue off to isolate rna. I then performed qRT-PCR and my gene of interest has good signal, so there is no RNase contamination of the slides. I am also using new bottles of reagents and try to be as clean as possible.
Any suggestions?
0
No replies to this topic
