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…

Great, thank you! Super helpful!

I am a colleague of achamess and adding some information about sample pretreatment. [image] june: For sample pretreatment, do you skip the hydrogen peroxide step prior to protease IV when using the v2 kit? We use both hydrogen peroxide (10min@RT) and proteaseIV (30min@RT) as pretreatment for…

Thank you @MGM and @achamess ! I am interested in costaining for my GOI and nociceptor markers (Trpv1 etc) to quantify the extent of colocalization. I know my GOI is expressed in the DRG from qPCR but have no sense of whether it’s a high or low expressor. Would you still recommend the v2 kit for this…

Costaining with IHC or another RNAscope probe? I’d use Cy3 for Trpv1 and Cy5 for your GOI because we’ve found that has the lowest autofluorescence. Cy3 is brighter but it may be hard to find real signal if your gene is a low expresser. You can get a sense of the expression of your gene from gene exp…

Do another RNAscope probe. IHC after ISH is tricky. Sometimes it works, sometimes it doesn’t.

Got it! Excited to try this out.

Hello again! I was curious whether section thickness makes a big difference in your hands?

We do 12-14 um. I wouldn’t do more than that.

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

Experimental Techniques

june August 16, 2018, 11:15pm 28

Sounds good, we will try that. I can do either IHC or another RNAscope probe–would you recommend one way over another?

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

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