Hi all, I’m a grad student trying to adapt the v1 RNAScope protocol my lab uses for adult mouse DRG to whole fresh-frozen spinal cords from P0-2 and P7-10 mice. None of us have worked with postnatal mice before, but my PI thinks I should be able to section the lumbar cord to visualize both the DRG and dorsal horn neurons.
Despite copious troubleshooting, I often lose much of my tissue during the experimental process. The bulk of section loss seems to happen during the 2 hour hybridization step, but I’ve noticed it as early as the EtOh dehydration.
I’ve called the company and they suggested (1) increasing the fixation time and (2) baking slides at 37-60C for 30-60 minutes. I’ve previously gleaned from lurking in this forum that (3) using thinner sections (14uM instead of 20uM), or (4) increasing the drying time after sectioning might help as well. Other than experimenting with these parameters, I always follow the original ACD protocol for fresh-frozen tissue, including use of Superfrost Plus slides.
I’ve manipulated all four aspects described above in a few different ways.
- I’ve increased the fixation time from 15 to 30 minutes, and then to 45 minutes. This alteration seemed to help the most.
- I’ve tried adding a pre-protease baking step at 37C for 30 minutes, 48C for 30 minutes, 48C for 45 minutes, and 55C for 30 minutes. Baking helps, but it’s unclear if using more time or a higher temperature is significantly better than the minimum.
- I’ve tried cutting 14uM sections once-this didn’t seem to help with retention at all.
- I’ve increased drying time from 30 minutes to 1-3 hours. I typically end up with a range of drying times based on when I finish making the slides; 2-3 hours is seemingly ideal.
Even with all the different permutations I’ve tried, I always lose at least half my sections before the end. There are also many different variables that I could alter that it’s hard to tell exactly what exactly is the best thing to change now.
If someone already knows a good procedure for whole postnatal cords, I’d love to hear about it.
If not, I’d still appreciate advice on how to develop a better procedure on my own. All of these lost sections are wasting us money, and I’m wary of overdoing a step and losing the signal completely.