My lab uses paraffin sections for in situ hybridization analysis all of the time with good results. I find that the morphology of paraffin-embedded tissue is much better than in cryosections. Once the paraffin is removed from the histological section via histoclear or xylene treatment followed by tissue rehydration through decreasing concentrations of ethanol, the in situ protocol is similar to cryosections. When embedding your tissue, make sure that all reagents (e.g., PBS, your fixative, ethanol, histoclear/xylene, etc.) are clean and RNase free. Good luck!

Hi Erika, thank you for answering! My lab is now doing ISH to detect virus RNA, 20 years ago they did ISH to detect mRNA.

I have now the same protocol, but it did not work (10 times) although my positive controls with astro virus detection is always positive. So I think the reagents are clean. I produced my RNA-probes (around 400bases) on myself and sequenced the corresponding DNA. It was the right basesequence. I tried different concentrations of my probe (like it is described in the roche protocol) and I tried different dilutions of proteinase K and I Pepsin. The last 8 times my positive controle (astro virus) for my actual positive control was every time positive. So I don't know how to go further. Do you think that SSC conentration (I have always 2x (and 0,2x), but in my hybridization mix 4x) does that make any difference, that would be my next plan to change it.

Thanks a lot, Daniela

Hi Daniela,

If I were you, I would use the exact conditions where your control probe works the best (e.g., proteinase K and pepsin concentrations). However, saying that, we use 1x SSC in our post-hyb wash solution (1X SSC, 50% Formamide, 0.1% Tween-20). Also, see file attached for the components of our hybridization buffer.

Since your probe is 400bp, you might consider doing an alkaline hydrolysis step on your probe, which may help probe penetration into your tissue. I typically would only do this on probes greater than 600bp, but it might help in your case. See attached file for alkaline hydrolysis protocol.

Although you might have addressed this already, make sure that you are using the anti-sense transcript. Since you should be using a sense probe as your control, you have probably already eliminated this as a problem. Also, double check that you are using the correct RNA polymerase at the correct temperature. Hopefully you are checking your in vitro transcription product on an agarose gel along the way to determine that the probe is present and not degraded. 1ul is sufficient.

Another problem might be that your probe concentration is too high. If this is the case, sometimes absence of staining will result or alternatively, non-specific staining. You could try a very low concentration and see if that works. We use 1:2000 dilution.

I hope that this can be of help to you. Good luck!

Erica

Hi Daniela, we do a few mRNA ISH on clinical specimens. (EBV, kappa, lambda)

Zytovision has a very good pretreatment kit for this purpose. I think it would be helpful to look at their website. http://www.zytovision.com/index.php/zytofast/33-zytofast-cish-introduction

good luck, Gudrun

Hi Daniela,

My main thought would be that your positive control is working fine suggesting the protocol you are following is good. Is the pos control also dog tissue? if so why would the buffer or proteinase K concentration be an issue for your unknown samples? If you are probing your pos control slides with the same probe as your samples, and both the control slides and sample slides are paraffin embedded i would think the issue would be the probes/target and not the protocol? It may also be worth considering another control probe to check that the tissue on the sample slides is viable and not degraded as that could be a factor i.e. current probe is fine but the target may have degraded?

Those are my thoughts,

Stephen 

Hi everybody,

thanks a lot for your suggestions! The positive control is also dog tissue, but the probe is 4 times shorter and for detection of virus-RNA. I am also doing IHC, but it don't work either. It is only published for working in rats, mice and humans. Maybe it is not enough mRNA, so you are right, Stephen... All samples are paraffin embedded, but I can't check my probe on the positive control slide, because there it is not expressed. That is only my positive control for my positive control.

Yes, I am using sense and antisense probe. I checked my sliced DNA out of the plasmid with gel electrophorese and my RNA-transcripts afterwards with dot blot.

I am now trying a lower salt concentration and maybe afterwards a bigger dilution.

Thanks for your help!!!!

Daniela

Hi everybody,

now my IHC is working for both molecules on dog tissue, so there must be enough protein. The question is, is there also enough mRNA for detection with ISH. I tried it with 1x SSC in the hybridization mix and with probe concentration of 1:25. There was again no staining.

As I said I made the digestion with pepsin and with proteinase K and I tried different probe concentration(1:2;1:5; 1:16). I controlled my probe with DNA-sequencing and afterwards dot blot with my RNA-probe.

What else can I change except probe concentration 1:2000 like Erika said to increase the staining?????

Thank you sooo much for your help!!!

See you, Daniela

Hi everybody,

Sorry if jump in the conversation. I am looking for suggestion: do you use all the solutions RNAase free? How do you with the paraffin? We have an histology department which process (embedding) all the samples together, so now I am concern that the embedding step could compromise my samples. Thank you.