We do RNA ISH on formalin fixed mouse brain slices (not paraffin embedded or frozen)(40 µm) with DIG-labelled RNA riboprobes as described in the attached file.
The problem is that the success of the protocol is about 40%. Once we got sign, then there is no signal, even if the circumstances are almost the same. May be the duration of perfusion with DEPC treated PB(± 2 min) and PFA-fixed brain slicing differ time to time, could it cause significant mRNA degradation in the tissue?
The other possibility we thought is the degradation of the probes. We store them at -20°C and dilute during ISH as described in the protocol (in the attached file). What is the best way to check the integrity and functionality of the probes? Or could be something else the problem?
We started using this technique recently, so we have not so much experience in RNA ISH. We appreciate every help!
Not my area of expertise, but i briefly worked with rat brains after electrode implantation... I found I had to use very freshly made parafornaldehyde to fix brains and not leave them for longer than 1.5 weeks in that solution. I have no idea if that matters..but am throwing it out there.
i use the ISH but on cryo section and it give me nice results. it differ from your protocol a little bit as i leave sections in th stain for one day, you can try it.
My experience concerning the ISH probe is that it is always better to keep them at -80C when they have been mixed with formamide. However generally when a probe degrades it is usually pretty homogenous and you should have nothing labeled and not some that are and other that are not. I think also that you could maybe increase a little bit the duration of the proteinase K permeabilisation step as you have sick sections.
Hi, I have done sectional ISH (100um) and I agree with previous answers. Fixation at 4°C and vibratome iced with pre-cooled PBST. A couple of my steps differ from your protocol...I pre-hybed in Hybe solution + tRNA and heparin (1:100 ea) for 2 hours, my protocol did not have the cool down step in between prehybe and hybe, I had a 15 min rnase treatment after your second wash step and I color developed for 1 - 4 days @ 4°C. I also use LNA probe, more stable. Hope any of this helps. Good luck!
Hey! I have performed Whole Mount ISH on zebrafish larvae and embryos for a long time. I have never experienced Probe degradation but an easy way and pretty straight forward way to check your probe is to run it in a normal electrophoresis on agarose gel. The only tip is to prepare your loading sample using denaturating loading buffer and heat it for 5' at 65 degrees before loading on gel and also stop the run shortly after the buffer leaves the well! You should be able to see a discrete band which is your probe. I use to have really high concentration like a range from 50 to 200 ng/ul! Another way to check probe integrity might be nano bit?? Then I used to store probes suspended in DEPC or Nuclease Free Water at -80 degrees OR at -20 but diluted in Hyb Mix. Also check the Ta of your probe! there's an algorithm to assess the best Ta for a probe considering % GC in your probe, %of Formamide and concentration of SSC in your Hyb Mix. Also I would suggest to try to lower a bit the Ta, as it might be too high! you have to find a compromise between being able to see the signal and having a bit of background staining! Also try and stain for longer. I used to stain O/N at 4 degrees sometimes. Let us know!
I would never immersion fix for longer than 12-24 hours in 4% paraformaldehyde. If storing sections for ISH, following sectioning on vibratome, sections should be cryoprotected (30% sucrose-polyethylene glycol) and stored at -20 (in cryoprotectant). Of course, all solutions, including cryoprotectant have to be treated with DEPC. In our experience this ensures that mRNA signal is well preserved for up to 3 months.
If it's 4% PFA overnight fixation, it should be at 4°C or only for a few hours at room T. I was working on whole 9.5 dpc mouse embryos and was transferring them to methanol for longer storage but if you just fix them with 4% PFA staining is usually better.
Probe itself plays a big role: some probes work right away and don't have any problems while you have to incubate others with NBT-BCIP longer. You can try it with a different probe, longer incubation time and higher antibody concentrations. Basically you can try and tweak every step of the protocol, for me it was always much easier to make a new probe than to spend a lot of time on checking every parameter.
- Badges
- Science topic
- Similar topics
- Biological Science
- Histology