Hello all,
I will be perfusing mice with PBS for brain harvest and a colleague would like to collect the blood via cardiac puncture (these are 18m old mice so would be nice to maximize use). Has anybody successfully done this? Or is it too late to get the heart to pump the PBS after blood is taken? Is there a good way to collect the blood as it pools otherwise?
On a separate note, i will be separating the hemispheres for 1) RNA (hoping to do 10x spatial transcriptomics) and 2) fixation for histology. Does anybody have any recommendations for preserving RNA quality during the PBS perfusion? Thanks!
Alexis
Hi Alexis
If I may ask, what do you intend to do with the blood? For cardiac perfusion with PBS, sure, you can collect the blood after you incise the right atrium, releasing the blood.
But depending on your flow rate, this blood will be draining out quite fast, so I am not sure how you will maximize collection efficiency this way....but if its only to collect small volume sample, go for it.
Slowing the PBS flow rate at first may be done but cannot exceed 30s-1min as that will disturb the overall fluidics and alter tissue characteristics.
What I will not suggest is to do a cardiac puncture to drain blood first, as its important to have the heart beating when starting the perfusion (at least we do it that way to avoid clogs during perfusion, especially for organs with fine vasculature).
With regard to RNA preservation during perfusion, it will be tricky. However, if you will be doing RNA-FISH, then you can do in fixed tissue and tissues can be fixed during perfusion....just allow the fixative to go in after the PBS.
Hope this helps
Aparajita
I can't really suggest much regarding the perfusion, but for the second part of your question about RNA quality, I think the most important thing you can do is make sure your perfusion solution is cold. Our lab collects mouse brain tissue for RNA experiments and we have found that a delay of a few minutes between euthanasia and dissection is acceptable for RNA quality (RIN 9+) so long as the heads/brains are cooled immediately on ice and dissection is conducted on a chilled surface. I would recommend using ice-cold perfusion solution and possibly placing crushed ice around the animal's head during the perfusion (after deep anaesthesia has been verified, of course.) Immediately after finishing perfusion, remove the brain onto a chilled metal platform to do your hemisphere dissection. Our lab uses stainless steel lunch boxes packed with an ice slurry and we cut on the bottom surface (with the lunch box flipped upside down). If you are using samples fresh, drop them into ice-cold PBS in tubes packed in crushed ice until ready to start your experiment. If you intend to freeze them down, snap-freeze them as quickly as you can. Hope this helps :)
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