Thanks for the advice Subrot... I am incubating with the hybridization buffer for 30 min-1 hour before hybridization, and I am doing the post-hybridization washes (2 times 50% Formamide 5X SSC, 2 times 0.2X SSC) at the hybridization temperature. I've also played around a bit with the proteinase K treatment, but I've found that if I treat much longer than a few minutes, my sections do not stick well to the slide. I will try your other suggestions though!

Hi Jaimee, try increasing the hybridization temperature to 5-10 degrees below Tm. This usually helps to get rid of aspecific probe binding... Good luck!

Hi there,

I typically work increasing stringency of the hybridization phase (either increasing salt concentration and/or increasing Temperature of hybridization) and keeping similar conditions in post-hyb. washes. Playing with post-hybridization salt concentration, and/or temperature ALONE in my experience is not enough, once LNAs are bound are bound...

Hope this helps, good luck, Ciao, D.

Hi Jaimee - what makes you think it is background? could be that your miRNA is expressed ubiquitously? also what do you wash your samples with after the colour reaction?

The miRNA I am studying is well known to be expressed only in epithelial tissue, but I am seeing expression in dermal tissue as well. After NBT-BCIP I wash 3x 10 minutes in 1x PBS.

Hi Jaimee, after the colour reaction I use 5xTBSTw to do the washes in. This stops the colour reaction completely and washes out all the background if you do not over-stain the tissue. Also it is worth doing multiple colour reaction / wash steps to ensure background does not build up.

Good luck.

Hi Jaimee, I've been trying to optimise conditions for specificity with a particular miRNA. As other people have mentioned increasing the hyb temperature to 32 degrees seemed to help but also washing at 52 degrees seemed to produce the optimal signal, even though this was quite close to the tm. Just by varying this by 4 degrees produced a quantitative difference so may be worth playing with.

Hopefully helpful.

Good luck.

Thanks everyone for the suggestions! I tried a variety of different things but in the end what worked best was doing the post-hyb washes at 2 degrees higher than hyb temp, as well as washing the secondary antibody off more stringently (3x 20 minutes in 1x PBS).