Cryostat Worries - I am trying to get good cryosections but am failing badly... (Oct/18/2005 )

Hi all,

I have some problems with making sections of mice brains with a cryostat. The rough story is as follows:

I am investigating a binding of a toxin to the CNS of mice, and I have injected a tagged toxin into the mouse brain. I exsanguinate, then remove the brain to place into dissection buffer (Tris-acetate, pH 7.4, 20% sucrose, EDTA and protease inhibitor cocktail). I cannot place them into 4% PFA in PBS, as there will be non-bound, cross-linked artefacts. So the brain is washed in dissection buffer, snap frozen and placed into OCT for cryosectioning. This is later fluorescently stained and detected under fluorescent confocal microscopy.

Problem:

The section structural integrity is lousy. No matter what thickness of the cut, they seem to lose their nice structural morphology. I would like to get my stuff into a good paper, so lousy quality pictures are no good for me!! Also, my boss thinks I am procrastinating because of it!!! HELP!!!

Any idea to improve the staining? I am using a secondary antibody with an alexa488 (FITC range) antibody.

THANKS!!!

I've had similar problems with cryosections of porcine cornea, since it is such a delicate tissue I've tried several methods for freezing. Snap frozen sections were by far the worst. The tissue integrity was more or less gone, collagenous structures appeared to be swollen and disrupted. Maybe this could be a problem in your cryosections too since they are not fixed in PFA.

In my case, cryoprotection with sucrose-solution (5 %, 1 h, 10 % 1 h, 15 % o/n) and "slow" freezing with Tissue-Tek in -80°C worked way better than snap freezing.

You might give it a shot!!!

hi
tek is a resin composed by polyvinyl alcohol 11% and carbowax 5%. I've used it for cutting rat brain.
I've got smarter results with bloc at -30° and knife at -20°.
Do you use gelatined lammelles to pick your sample?