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…

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 mRN…

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 i…

Hello. Thanks for your question. You still need different channel probes if you want to multiplex. The HRP steps are still based on channels.

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…

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 li…

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 circ…

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 t…

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.

Did anyone try the Molecular Instruments HCR RNA-FISH? [image] Molecular Instruments, Inc. | In Situ Hybridization + Immunohistochemistry Hybridization chain reaction (HCR) imaging technologies open a new era for in situ hybridization (ISH) and immunohistochemistry (IHC), enabling simult…

It’s been sitting in my freezer waiting to be used. Thanks for the reminder. It looks very promising. Have you tried?

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

Experimental Techniques

achamess July 17, 2022, 10:04pm 88

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

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

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