In situ hybridization is a powerful method to interrogate gene expression in tissues. It is known as being a challenging procedure due to the many steps where errors and obstacles arise. We’ve recently begun to use a commercial system called RNAscope from ACD Bio that gets around many of the pitfall…

Our lab has also used RNAscope, but on dissociated mouse DRG neurons that were fixed with 4% PFA /4% sucrose and stored for up to 1 year at 4 degrees. As was mentioned, the assay is very straightforward, and I’ve gotten really nice, clean signal for my target probes. However, I am struggling with ho…

Hi @TSheahan . Thanks so much for joining the forum and for your question. Quantification is definitely a tricky subject, one that I don’t have a perfect answer for. But the answer will depend in part on what you’re trying to quantify. If you’re looking for a binary answer (express/not expressed) t…

Another response on Twitter [image] Elizabeth Sypek, PhD (ebethsypek) @achamess I just set a threshold, specific to the gene of interest (bc expression levels vary so much, 4 puncta can be + for one but not another) Alex Chamessian, MD PhD @achamess RNAscope (In situ hybridization) to st…

Thanks @achamess ! Just enrolled in a very timely webinar with RNAscope on data analysis and representation. I’m interested to hear their recommendations as well. http://rna.acdbio.com/SupportWebinar_Jul25?utm_source=acd&utm_campaign=support_webinar_07_2017&utm_medium=acd_email&utm_term=register&utm…

I’ve recently completed some successful RNAscope experiments (with triple label IHC) on fresh frozen lumbar DRGs and spinal cord. Tissues were collected, frozen on dry ice (in OCT), sectioned immediately at 20um on a cryostat (onto the slide), dehydrated in EtOH, and fixed in 10% formalin for 15 mi…

Hi @sshiers Thanks for the post and for sharing your tips. You’re absolutely right about the protease time and fixation time being critical variables. You used fresh frozen, which they have a protocol for, although it seems you’ve reduced the protease time substantially. I actually apply the fresh…

Mouse or rat? Yes, I shortened the protease time due to my short fixation period as I didn’t want to disrupt my IHC label. I was trying to find that sweet spot between too much fix and too much protease. :smile: Have you tried a shorter perfusion with shorter protease time? Could cut down on your …

Hi @sshiers That’s 2 hour post-fixation (piece of cord in PFA in a tube @ 4C), not perfusion. That’d be an insanely long time and I’d be in grad school forever :slight_smile: I perfuse for 3-5 minutes with PFA, take out the cord or DRG, the do the post-fix.

Gah read your message wrong. Phew :slight_smile:

@esypek Hey, have you used the v2 kit with three colors, including Fluorescein? Cy3 and C5 look awesome, but Fluorescein is garbage. The issue is definitely the fluor, since we can use the same probe, same tissue, and get wildly different results. Maybe our fluorescein is bad? Green is never my fa…

RNAscope (In situ hybridization) to study gene expression in pain-related tissues

Experimental Techniques

achamess July 6, 2017, 6:04pm 4

Another response on Twitter

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RNAscope (In situ hybridization) to study gene expression in pain-related tissues

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