Drop (immersion) fixation vs. transcardial perfusion of DRG and Spinal Cord


#1

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.

Thoughts? @liz @tberta @vanja


#2

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


#3

Thanks @vanja. That’s encouraging, and consistent with my past experience too.


#4

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.


#5

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#7

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!