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.
Hello All,
I was reading through this great information and was hoping that this thread was still active, or had users who would see my question.
Below are some representative images of a multiplex RNAscope V2 kit I ran with 4 channels. C1-opal dye 520, C2-opal dye 570, C3-opal dye 690 with a 1:1000 dilution factor. The C1 came in prediluted we diluted the channels 50:1:1. Then dapi was added at the end. I followed the proctol to the letter. Every channel looks good except my C1. I know others have mentioned the greens not working as well.
Do you all recommend I change the dilution for the 520 opal dye? How would you properly quantify the green channel? I have been selecting the weaker green signal and using it as background subtraction in Fiji, and then counting. I’m just unsure of my counts because they’re usually not clear puncta, like the other channels. I am hoping that is subjective enough. You can see something similar in C-3, but it’s not as bad. Would you only count clear puncta? Thoughts?
The images below are all the same sample:
