I was recently told by the director of our pathology core that brain tissue must not be perfused if it is to be frozen (to be later sectioned via cryostat). She said it would lead to shattering of the tissue because of the introduction of liquids. Instead, she recommended snap freezing fresh tissue with something like liquid nitrogen.
However, the only way I worked with brain tissue in the past was transcardial perfusion with 4% PFA, postfixing in PFA, embedding in OCT, then snap freezing over dry ice. I never seemed to have any problems with tissue integrity.
Could someone offer their opinion on fixing brain tissue that will be frozen later? How do you observe the tissue responding?
I always perfuse the brain with PBS first and then with PFA4% and then embedded it in OCT. Some people do not fix the tissue before the embedding they do it during the staining normally with acetone. But it always depend in the application...
I think this might be some misunderstanding with your director. Many people formalin-perfuse the brain before dissecting and freezing. Perfusion in itself would not be expected to cause freezing artifacts, and it tends to make the brain a little less prone to dissection/handling damage than a fresh brain. As for the freezing, she is correct that snap-freezing using liquid nitrogen (using an intermediate bath containing isopentane) is usually preferable to freezing over dry ice, because the temperature is colder and more constant. However many people use dry ice, as it is acceptable and easier to procure than liquid nitrogen, and if done properly the difference in freezing is probably marginal.
You could equilibrate the tissue in a sucrose solution for cryoprotection before freezing the tissue.
I agree with all of the respondents. I have performed many a mouse perfusion (PBS followed by 4%PFA) with the specific intent of sectioning the brain using a cryostat.
Best.
Hi Nathan, the important thing is kryoprotection after PFA/Formaldehyd fixation to prevent kryoartefacts. After fixation put your piece of brain tissue in 30% sucrose and wait until it has been fallen on the ground of the tissue container. Depending on the size it will take 2 - 7 days. Then you can deep freez it (for storage -80°C over isopentan in dry ice or liquid nitrogen) or freez it directly in the crystate and cut it.
Good luck!
You can
1. make thick slices (e.g. 100mirometer) of raw brain by microslicer or vibratome, and you can proceed to any further processing procedures.
2. make very thick slices (e.g. 2mm) of raw brain by a knife, put in OCT compound and make cryosections, collect them on coated glass slides immediately put in some fixatives. You can proceed to any further processing procedures.
Usuda N
We always perfuse and fix by injecting first 10ml PBS in heart then 4 percent PFA I take the sample and put it in sucrose 30% for two days after that simply place tissue in OCT block and fill it with OCT no shattering occurred so far
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