What do you all think about immersion fixation vs. transcardial perfusion for certain tissues? I realize for large tissues such as brain, perfusion is the right way to go, since you can ensure that the core areas get fixed as much as the surface. But for really small tissues like DRG, and even spinal cord, immersion fixations seems as if it would be just as good. In fact, when I’m lazy, I do immersion fixation of DRG and SC, with 2 hour fixation at 4C, and the results seem as good, if not better. This is for standard IHC stuff.
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Hi there - We routinely only post-fix DRGs overnight (for some sensitive antibodies only for 10-20 minutes, we never perfuse for DRGs. Additionally, depending on the quality of the antibody we also skip the perfusion step for brain, postfix overnight, cryo-protect, freeze and slice without any noticable difference. Cheers, Vanja
Thanks @vanja. That’s encouraging, and consistent with my past experience too.
Update: I tried drop fixation for some DRGs from a nice transgenic animal with GFP in it. The tissue looked awesome and the fluorescence was better than usual.
We did RNAscope today on the tissue, and the signal was beautiful. I fixed for 2 hours at RT.
So, for DRG, perfusion is not necessary for most applications. Perhaps if you’re needed to stop a biochemical reaction (pERK?) or wash out blood cells, perfusion would still be advisable.
Thanks @vanja for the encouragement.
Just agreeing with above – I’ve done hydraulic extrusion and drop immersion for SC with great results…also, doesn’t hurt that it saves a bit of time!
Please I would like to know the differences between immersion and perfusion fixation in tissue preparation. Thanks!
Drop means you take fresh tissue and just place it in PFA and let it sit. The advantage is its very easy and you don’t need to do perfusion, which can be challenging and messy. The potential disadvantage is that it takes time for PFA to perfuse from the outer surface of a tissue piece to the inner core. If you have a large sample, like a rodent brain, this will affect your results. Outer layers will be fixed faster and longer than inner core structures. This will inevitably lead to variation based on location.
Thus, for brain, most people prefer to perfuse. For spinal cord and DRG, I think immersion fixation is adequate because the tissue is small.
Hello, how long would you recommend fixing the spinal cord for IHC? I’ve fixed DRGs for 2 hours at 4C but was wondering if the cord would need more time. Also would you have any tips on how to dissect out the spinal cord? Thanks!
2 hours as well for spinal cord.
For dissection, see:
https://forum.painresearcher.net/t/mouse-spinal-cord-and-drg-dissection-video-active-users-only/196
This is perfect, thanks!
I have a follow up question on this.
What is the next step from PFA fixed DRG if you want to embed in paraffin? I’ve seen multiple protocols mostly with 20-30% sucrose overnight 4C.
Coming late to this sorry, I tend to to a combination of both. I do a perfusion and then let the tissue sit in 4% PFA usually over night. I find this particularly useful for tissues I find hard to extract in non-perfused animals…DRG’s, Trigeminal Ganglia etc.
Any tips on how to remove these from non perused animals would be a big help!