Dear Angel del Marco

Check this one:

https://www.feinberg.northwestern.edu/research/docs/cores/mhpl/tissuefreezing.pdf

In my old lab after fixation, we would then put the brains in 15% sucrose overnight, followed by overnight in 30% sucrose, rather than straight into 30%. This works well for us, and I have tried this with brains that were not fixed by perfusion and it still worked pretty well.

Hi Angel,

I would suggest two points based on your question. But before that you have to keep in mind that although PFA is considered the ideal fixative for most of histology and immunohistochemistry procedures, it is very slow fixative (except for biopsies which needs just few hours, it usually takes several days up to 2 weeks to properly fix a piece of tissue). Also PFA can not penetrate tissues effectively (under ideal conditions, a 0.5 cm3 cube would take ~ 2.5 hours to be intially permeated by formalin, in other words to just stop autolysis and keep the tissue intact). Discoloration of tissue after keeping it for few hours in PFA/formalin is an indicator that fixation is ongoing, but you have to wait for some time to allow complete crosslinking of the proteins, don't worry about exceeding the optimal duration as theoretically, there is no overfixation. You have to only worry about under fixation or the incomplete fixation. Thus, perfect fixation requires both PFA concentration to be maintained at least 4% and the duration should be not less than a week provided that the organ/tissue is cut into small pieces.

The brain is very peculiar organ as it contains very high amount of fluid. If you start fixing this organ in 4% PFA or its equivalent 10% formalin, the concentration will substantially decrease due to the fluids inside the brain tissue will dilute the fixative and ruins it. Thus, the fixative will function in an imperfect way.

Another thing to consider is tissue dehydration prior to embedding in OCT. Gradient dehydration will give you almost perfect tissue structure. Going to a very high concentration of Sucrose will adversely further affect the tissue architecture.

My two suggestions for you:

1. Slice the brain as small as possible and fix it in 8% PFA (=20 % methanol free formalin). Personally, I prefer using the neutral buffer formalin to the PFA. You have to change the fixative every couple of days till complete hardening of the tissue. Some people also fix the brain as a whole in 50% formalin for one week, then re-fix in 10% formalin for few days. It is up to you to choose the best one fitting your work. Please consider fixing the tissue as soon as you collect it. This point is much more important than the concentration of the fixative it self. If you are collecting from several mice, plan and do one by one.

2. Dehydrate in three concentrations of Sucrose: 10, 20 and 30%. Keep the tissue cassettes in these three solutions sequentially for 2 hours for the first two and over night for the last one. The time can be optimized. A good sign for you is that the tissue sink down to the bottom of the falcon tube. Make sure to use a container that wide and tall enough to allow the tissue to sink.

Best of luck

Ahmed

Thanks for your quick answers.

Prof. Gheita , thanks for the link about freezing methods and pros&cons of every technique. We did freezing with liquid nitrogen and we got an "unpredictable pattern", as the link says. It has been so illustrative.

Amy Lloyd , thanks for your tip about the sucrose gradient for cryopreservation.

Ahmed Abdellatif , thanks a lot for your complete explication and for your time in it. I will follow all your suggestions, I think that they are so useful for our problem and we could solve it with them.

Thanks again!

Best,

Ángel