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 pitfalls of traditional ISH.
I’ve used it a lot now in the spinal cord and DRG. Below is an image of the gene VGAT (Slc32a1) in the dorsal spinal cord. There is nice expression, and most importantly, it was a quick 5 hour protocol for results. I purchased the probe, performed the procedure (with some modifications) and then got the data I needed.
Downsides are that the reagents are expensive (although the time savings make it worth it), and that there still needs to be some optimization with respect to tissue preparation. It is this last step that I think is worth discussing here.
In our lab, we’ve settled on using the Fresh Frozen Tissue Preparation procedure, even though we use fixed frozen tissue. We perfuse our mice with fresh 4% PFA, then post-fix for 2 hours. 30% sucrose overnight (RNase-free) and then slice at 14 um onto superfrost plus slides. Then we use take the sections forward using the Fresh Frozen protocol, as opposed to the one from ACD that is indicated for Fixed Frozen. In addition, we skip the initial 15 min post-fix in the Fresh Frozen protocol. Everything else after that stays the same.
If you’ve used RNAscope, please share your experiences and whatever tips/tricks you’ve picked up.