Hey. We haven’t tried and it’s not high on the list right now. But if you do it, please let us know how it goes. Good luck!
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Hey Alex, any new info on that. I’m holding on buying them until I get reliable feedback. Hope it’s all good at your end 
Sorry. We haven’t done it, and honestly, probably won’t anytime soon. There is a lot of stuff in the queue. I asked Twitter if anyone tried but didn’t get any responses. I do see AIBS and Janelia people using it though, so I bet it works. Good luck!
I finally tried a single labeling. At first, the signal looked weak, but in our microscopes alexa fluorophores work a lot better than OPALS so I would say I’m happy with HCP to begin with. Now I’ll move on with multiple probes.
The big disadvantage to me is the timing as the intervals for reactions can be quite annoying to fit reasonably in a schedule. Of course the main pro is the cost-yield ratio.
An example of my first trial in DRG using Calca as probe:
Here’s a tiled acquisition where the background is more evident.
Hope this to be helpful for those hesitating like I was…
That’s beautiful! Thank you! I’m convinced. Thank you so much for sharing.
So do you plan to now go ahead with this over RNAscope?
What do you mean about reaction schedules? Too long? Maybe there is a way it could be automated.
I know Allen Institute uses this for high plex FISH.
Thanks!
Yes, the plan is to move to HCR when multiplexing turns our imaging with OPALS impossible.
As for your second question, two incubation times of 12 to 16 h can be tricky to organize specially when the time between them is short. I’ll try to have one of these incubation shortened as much as I possibly can.
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Just an update. Molecular Instruments, which makes HCR, has sponsored a post here and is offering a free starter kit. For those interested, definitely check it out and let us know how it goes. @Joyce_MI
Hi achamess,
I have through these RNAScope posts and I must say that I have learned a lot and appreciate everyone’s comments and experiences.
I am new to this technique and having gone through the posts it appears that what works is using fixed frozen tissues but following the fresh frozen protocol.
I was wondering if anyone has used the fresh frozen protocol from start to finish:
- how did it go?
- how did you preserve the tissues for long term storage?
I am working on mouse brain and I intend to cryosection at 12um which will leave me with a lot of tissues that would need to be stored for long, so I am worried about fresh frozen tissue integrity. I am a bit skeptical about using the fixed frozen protocol because I am worried the formalin may interfere with the result. - Lastly, why did you choose fixed frozen tissue instead of fresh frozen?
Has anyone used RNAlater? or how do you ensure your samples and solutions are RNAse free?
I would appreciate everyone’s thoughts and ideas on this.
Thank you.