I am trying to visualise the expression of a number of genes (including Arg1, Nr1d2, Per1 and Per2) in the liver (sections cut at 18 microns). However, I do not get any staining.
I know the protocol/solutions works because I use it routinely for brain sections and whole mounts. (My stringency washes after hybridisation step are: 5X SSC wash and 3 0.2X SSC washes). The probes work because I have used them in the brain.
I have tried reducing the hybridisation step to 40C and soaking the tissue in methanol overnight.
I am currently testing an increase in proteinase K digestion.
If anyone has any suggestions or advice I would be so grateful. Many thanks
After your washes with SSC, are you doing a set of formamide and salt washes?
Hi Leslie,
No, I previously used solution X (50% Formamide/ 2X SSC/1% SDS) but recently switched to SSC only washes as it worked better for my brain sections.
I have just tried going back to the formamide/SSC washes and waiting for the results this afternoon......
Hi Michelle,
The only other issue I could think of is that there might be a problem with the antibody to your developing solution. When I was having issues with my brain sections, most of the times I didn't get good staining was when there was a problem with the antibody or with my stains themselves.
Hi Michelle,
some time ago I used to do ISH on liver sections. It worked fine for all kind of genes, even for the less expressed. I used 10 um sections of fresh tissue, (but you can use PFA fixed, sucrose soaked - be careful for overfixation with the liver!) and hybriidization on 60°C. After all these years with ISH, I came to conclusion, that every tissue requires its own protocol. Check mine (in your mailbox). B.
Hi Leslie,
My antibody is working fine because I am using it for brain sections.
Switching to SSC/formamide stringency washes has not worked!
The biggest caveat of RNA ISH is the stability of RNA.
Cross-linking additive fixatives can also contribute to a) making an RNA inaccessible for hybridization and b) additionally fragmenting it. Also, if RNAs are small and overdigested, they can be nicely washed away in washing steps.
I will decrease the tissue thickness to even 6um. 18 um is way too thick. You might be unable to hybridize because of underdigestion...
Cheers,
Ivan
Hi Ivan,
Thank-you for all the tips! I will definitely be trying some of these!
Michelle
Go down to 10um at least. Best results I obtained with the DAKO Genepoint kit and protocol.
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