Thank you!
Yes, using mice DRG.! I used Opal 520 for NeuN, in case you would like to see. I’m not sure how good these images are. mCh_B_20x_Lospeed_052919_Airyscan%20Processing_NEUN_DAPI%20copy|500x500
Is that Rbfox3 probe or antibody?
NeuN (Rbfox3) isn’t very highly expressed at the protein level in DRG in my hands.
I see some signal there but I’d use something else as your positive control. Not sure what your target of interest is but I would step back and confirm that your system works. Use something like CGRP, which is well expressed in DRG (see above). Or you could use the RNAscope positive control probe. Nav1.7/Nav1.8 is also very high expressing by RNAscope.
See the post above for protease:
I think you might be fixing too much. Try 2 hours for DRG.
Thank you!
I will try your suggestions and see if I have better luck.
Hi @achamess
First, I’d like to say thanks for all the info you’ve contributed to this discussion, it really helped me to get the protocol up and running.
Secondly, although you don’t recommend it, I wonder if you have more specific wisdom to impart regarding RNAscope followed by IHC. Through my attempts to combine these protocols I have managed to get very clear ISH signal but the IHC signal is noisy, even for antibodies that I know stain my tissue beautifully normally.
Some background: I am using 30um mounted mouse spinal cord tissue and following your adapted fresh protocol for RNAscope followed by a pretty standard IHC protocol. I’d appreciate hearing about any adaptations that you have tried.
Thanks @clarereynell. So glad the discussion here helped you.
I don’t have much to say about IHC after RNAscope except that your description fits my experience too. It looks ugly most of the time. It’s related to the protease treatment I suspect, and if you want to get your IHC to work, you may have to fiddle with the protease pre-treatment. That’s all I can say. I know people do IHC after RNAscope but it seems very antibody-dependent. And in my hands, there is always more background, even when it works.
Some things that may help:
https://acdbio.com/science/applications/research-solutions/dual-ish-and-ihc
If you can answer your questions using just RNAscope, I’d do that. Alternatively, you can try a transgenic animal with GFP. That is the only thing that has worked for me.
Here is an example from a recent paper:
That is TrkB RNAscope and anti-GFP antibody targeting ChR2-EYFP.
The anitbody is: chicken anti- GFP (1:1000, Abcam, 13970, RRID:AB_300798)
Good luck. Keep us posted! Thanks for joining the forum.
@achamess This is an incredibly helpful thread! I just ran some RNAScope in spinal cord sections and it worked fairly well but I have a lot of background/autofluoresence in the green channel. I used fresh frozen spinal cords, 16um sections, and the v1 multiplex kit.
Based on this thread, I’m thinking of trying a few things: (1) decreasing the protease IV time (I’ll need to run some controls to figure out the optimal time); (2) using the Multiplex v2 kit; and/or (3) using fixed frozen tissue instead of fresh frozen.
For fixed spinal cords, do you perfuse and then post-fix for 2hrs at 4 degrees or overnight?
Glad you’re finding this thread and site helpful.
Green channel will always have the highest autofluorescence. Must you use the green channel? if so, pick the gene with the highest expected expression, that way you don’t need to crank up the light/exposure. This is what ACD recommends.
But even with that, v1 just isn’t as sensitive. v2 was a huge advance for me. I used to battle autofluorescence a lot.
My recommendation would be (in order):
- Get the v2 kit. It’s worth it.
- If you use green, make sure it’s the highest expressing gene in your panel
As far as fixed vs frozen, I use lightly fixed (perfuse, 2hr post-fix in PFA) and then use the fresh-frozen pretreat protocol (without doing the fixation step, since it’s already fixed). This works very well.
Good luck! Let us know how it goes.
Thanks for the recommendations. I’ll try out v2 with lightly fixed tissue and let you know how it goes!
I always dislike using the green channel but I’d like to colocalize 3 transcripts for these experiments. I try to keep it to the high expressors but sometimes it’s hard to know in advance!
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Follow up to this post.
ACD has introduced some kind of ancillary kit to facilitate IHC w/ ISH.
Looks like they do antibody binding before ISH probe binding. The kit looks like some blocking and diluent reagents. I bet one could use homemade reagents. The interesting thing is the workflow. The mRNA withstands O/N primary antibody incubation. Probably still would want to start with fixed tissue so that the mRNA gets some protection. They use FFPE in their protocol. But then they fix again after antibody staining, presumably to lock-down the Ab to the tissue.
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Hi,
May sounds stupid and very basic, but I’m pretty confused with this channel thing. There’s the channel of the probe C1 to C4 and of course the microscopy channels (Fitc, Dapi, etc…).
If I’m using v2, do I must have probes of different channels? Can’t I, for example, use probes designed to C1 in my multiplex assay? ACD makes probe channels appear linked the color channels, which is not really correct. Or did I misunderstand at all?
Thanks in advance!
Hello. Thanks for your question. You still need different channel probes if you want to multiplex. The HRP steps are still based on channels.
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Hello all,
What an awesome forum for spinal cord and DRG researchers! First, thank you for creating an environment like this one. I’ve already found it useful for my own work.
Our lab is new to RNAScope and I am currently testing how well the original protocol labels our transcripts of interest in the spinal cord before and after a contusion injury. So far the only modification we’ve made is to compare a makeshift incubator with the (what my colleague likes to call) EZ bake oven. So far we haven’t seen major differences, but this has been round 1. 2 and 3 will be done by (hopefully) the end of the week! Yay for science.
Our parameters:
Fixed frozen thoracic spinal cord tissue (injured and uninjured). We perfuse with 4% PFA in 0.1 M phosphate buffer (I’ve noticed others use PBS). Once we’ve perfused the animal we post-fix for 1 hr at RT and transfer to 30% sucrose. We then cut fixed-frozen tissue at 20 um. For some reason our lab has difficulty cutting any lower.
For RNAScope we are using the multiplex v2 assay and our goal is to be able to label 4 transcripts + DAPI. I understand this may be a tough task to accomplish so we’re keeping our options open, and are ok with limiting to 2 or 3. We’d also like to (eventually) perform IHC alongside.
We’ve gone through the complete protocol with only the positive and negative controls and had success with labeling. However, like many of you, the green channel is not great and I mistakenly paired it with the polr2a positive control (lowest expression).
If I understand correctly, the modifications made by some of you is to skip the post-fix 4% PFA for 15 min @ 4C, and antigen retrieval steps completely, and to use the prot IV rather than prot III. Do you still incubate for 30 mins?
I will continue to keep you posted about our progress and any modifications we make!
Thanks,
Michael
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Hi @Mdfors01. Thanks for joining the forum.
Sounds like you’re on the right track. I find the ‘positive’ controls from RNAscope to not be the best for neural tissue. They’re there, but not as strongly as other neural-specific genes. In any case, if you got the positive-control(s) to work, you’ll likely get your gene of interest.
Re: modified protocol
Yes, I do the fresh-frozen protocol on lightly fixed tissue, with the omission of the 4% PFA pre-treat (since the tissue is already fixed)
We start the protocol at p24 at the “Dehydrate the sections” step. We follow it exactly, using protease IV.
We find we can circumvent the antigen retrieval in this way with excellent results.
I do recommend trying to slice the tissue at 12-14 um.
Good luck! Keep us posted.
Did my first try in our new lab! Super thanks for all recommendations here, Perfused mouse > post fix max 2h > 30% sucrose > then, fresh-frozen protocol from dehydration step, using protease IV. I’m quite happy for this very first try. There’s a lot to improve, but I’m quite satisfied under the circumstances this was done. Using V2 kit with OPALS 690 (in red) and 620 (in green).- didn’t get the other ones in time so I used these anyway.
Love it! Thank you for sharing @defaria! Looks fantastic. Just curious, what would you improve? I see very little background. Strong signal. Good luck with all your future experiments. I love that this forum’s discussion was able to help you get satisfactory results on the first shot. Congrats on the new lab too.
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My microscope filters were not optimal, and I got some background from the OPAL 690 (not visible in this image, but present). I’ll share my next round with better settings… But, thanks to you @achamess, I saved a lot of time and reagents by following your modifications.
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Did anyone try the Molecular Instruments HCR RNA-FISH?
I’m curious about it, I heard it is a lot cheaper than ACD but not as good signal. Please, give me some feedback if you have ever tried.
Regards!
It’s been sitting in my freezer waiting to be used. Thanks for the reminder. It looks very promising. Have you tried?
We are ordering, but I’d love to get feedback from someone that has tried. It’s been three weeks, have you tried? 