I was trying to post fix the whole mouse brain in 4% formaldehyde and I was wondering what is the best time period I should suspend the whole brain in the solution so that the whole tissue gets fixed properly with minimal degradation from inside. Can anybody make any suggestion ?
Hello Sharif,
first of all you should tell us what you want to do after the fixation?
Paraffine embedding? Cryoprotection in sucrose solution and then freezing?
Usually I perfuse a mouse transcardially with 5 ml 0.9% NaCL (to get the blood out, you have also to open the right heart atrium prior injection), followed by ice cold 50 ml of 3.7% paraformaldehyde (PFA). I then postfix the brain in 3.7% PFA over night at 8° C. Then the brain is well fixed and you can continue with the procedure of your choice (see above, paraffine or freezing).
I suppose that fixation for about 24 h at room temperature will surely fix a non-perfused mouse brain very well either.
Hope this helps you.
Stefan
Thank you so much Stefan. You are correct, it is for paraffin embedding!
Actually I was trying trans-cardiac perfusion but something went wrong! And I was not sure if I tried post fixation of the brain whether I should leave it in Formalin for usual 8 hours or for 24 hours!
Thank you so much for your answer. Another issue is as in post-fixation the formalin enters the brain through diffusion process and the brain starts to degrade also from inside; Should I cut the brain in two halves for better perfusion with formaldehyde?
Dear Sharif, we usually cut the brain (rat brain) in 2 halves, clearly perfusion will be better like this. 12 h fixation could be enough for your purpose then. E.g. when we stain for lacZ activity, I try to keep the time as short as possible as this also diminishes the lacZ activity.
Just FYI, this also depends on the age of the tissue. Embryonic fixation, depending on the stage, should not exceed 8 hours post-fixation for use with some antibodies. Adult tissue will hold up fine overnight after perfusion, but I wouldn't expect to properly fix a brain only by immersion unless you're doing it for an animal younger than P5.
Dear Sharif, after transcardial perfusion we cut the brain (rat, mouse) in three pieces at the chiasma opticum and in front of the cerebellum. We perform post-fixation over night at 4°C and we always use fresh prepared PBS-buffered 4% paraformaldehyd. Good luck!
Andrea
- Badges
- Science topic
- Similar topics
- Biological Science
- Cell Biology