I've heard that their in situ hybridization system is really good, but in the website they say it requires the purchase of their hybridization oven (what they called HybEZ oven). I'd love to know if someone has tested their system and if it really requires the use of their oven (which is not that cheap, unfortunately).
Thanks !
I've used the technology. It is excellent. I used a water bath instead of the HybEZ oven.
Hi Natasha, I'm also interested in the technology and try to find a way to prevent to by the HybEZ oven. How do you exactly put your sildes into the waterbath? Our lab has an oven without humidity feature and I had the idea to use this and put some water in a bowl beside the sildes to increase the humidity inside the oven. Do you think this would be enough? Best wishes, Oliver
I put my slides in a sterile petri dish and lined the inner perimeter with moist kimwipes. The humidity is important. The petri dish sits immersed in the water bath, but not actually floating.
Could anyone elaborate on how they avoided purchasing the oven? Routinely in our lab we line the insides of a container with cotton and wet it with sterile water. This container is placed in our incubator which is set at 37 degrees. Does the RNAScope technology require a different incubation temperature? If so then I will have to use a water bath, but I am not understanding how the petri dish is not afloat and completely sealed if submerged. Any help would be deeply appreciated. Thanks!
I used water bath and it worked perfectly! I put my slide in petri dish, as Natasha mentioned, together with wet filter papers. Then I put it in plastic bag. By placing another petri dish below and a heavy ring on main petri dish I kept it in middle on water bath!
Another point about RNAscope: deparafinization step is very very critical!!!
Hope it helps.
I was able to get a loaner hyb oven from them, and ran a side-by-side comparison of their oven and our (non-humidified) oven. For our lab oven, I placed slides in a sealed plastic box lines with moist paper towels (slides were elevated on a plastic stand so they did not contact the paper). Both ovens were set to 40degC. Performance was identical from the 2 setups. I've only run this once so far though, your results may vary. As with any ISH, it's essential to keep the sections humid, but there's probably several ways to achieve that.
I actually bought the oven because I was not happy with the options in my lab. Found the oven really well designed for the task.
Probably I will use this techniques in future.
Can you recommend some papers on the technique.
Thank you
Manufacturer's instructions are clear and simple. Support is excellent from ACD.
We end up buying the oven after all. Not regretting so far !
ACDbio has some publications on their website I think, if you're interested in seeing some protocols.
We are looking at getting an Autostainer for IHC and was pleased to discovered that it has been optimised to do RNAScope as well.
I was wondering if there is any harm to incubating your slides with the probe in the oven (hybridization) overnight as opposed to 2 hrs that the user manual recommends?
RNAScope is very time and temperature critical. Follow instructions to the letter. There is a step when you can wait overnight so the workflow is done over two days.
1. Nope, we got results without HybEZ too. However later we got it to make life less difficult.
2. Another thing is best stooping point is hybridization and then storage overnight in SSC (see protocol). We dissected whole protocol workflow in three days ans still got fantastic results with animal and plant tissue sections.
3. Only change is after fixing bake samples for 30 minutes for better adhesion on slides.
Hi Shyam Solanki , what are your stopping points for days 1 and 2? I keep my slides in SSC overnight after 2 hr hybridization which is my stopping point on Day1 and then do the rest of the protocol on the second day, so mine is a 2-day protocol.
Ishita,
1st day - Sectioning, and fixing, storage in EtoH.
2nd day - Pre-treatments, Hybridization and storage in SSC.
3rd day - Post hybridization steps, and slide mounting.
It makes life more easier, specially with V2 process which takes additional two hours compare to chromogenic assay.
We are hoping to purchase an autostainer unit for our histology lab and some of these have optimised programs developed with ACD to do the processing steps for you. Either way RNAScope is brilliant and the technical support offered by the company is exceptional. I really recommend this product. We also break up into multi days for processing. The protocol states when breaks can occur but others have described above. RNAScope has worked really well on old histology samples that were processed without any thought of ever doing RNA work. Very robust system.
The HybEZ oven is only a temperature controlled environment so any precise incubator would substitute but temp is critical.
Terry Pinfold, how long were the "old histology samples" that you used fixed in formalin for?
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