RNase inactivation during IHC prior to LCM - (Dec/17/2012 )

Hi!
I am currently working to optimize a protocol for detection of specific cells in the intestine aiming at a strong specific signal and a high RNA quality. After IHC the cells must be captured individually using LCM and then analyzed for gene expression levels. I work with fresh-frozen tissue, stored at -80.
The RNA quality is initially good (not perfect but OK), but after (cryo) cutting, fixation, immunostaining and contrastaining the quality is very poor. I have optimized the IHC protocol to be quite fast (only around 20 min in aquous solutions), and I have added enzymatic RNase inhibitors to all aquous steps. As this doesn't seem to be enough, I am currently looking for efficient chemical RNase inhibitors. I believe (at least part of) the problem is with endogene RNase's, so the inhibitor must be able to penetrate the tissue (8µm thick). I have been recommended n-ethyl maleimide (NEM), but have not been able to find any information about how to use this.
So: Any good advice in general?
Any good ideas about chemical RNase inhibitors?
Any ideas about how to use NEM as an RNase inhibitor?
Thanks!

After fixation and permeabilization anything can penetrate the tissue.

I would suggest trying to do everything on ice if you are not already. What is your RNA extraction method post-IHC?

Curtis on Mon Dec 24 07:03:38 2012 said:

I would have thought so too, but even the addition of 3U RNAsin/µl ab dilution ect. does not seem to preserve my RNA. So I believe I have a lot of endogene RNases that just does not get inhibited fast enough:-(

Myworkismyplay on Thu Dec 27 04:06:37 2012 said:

Thank you for your suggestions! I have performed the IHC on ice (or actually in the fridge w. ice cold reagents), and it doesn't help. I purify my RNA with a column kit (Arcturus PicoPure) that I know works well, and always include a control which is fine.