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