Hi all,
Trying to section e14.5 mouse cortex and keep getting these tears along the lateral ventricles. I'm trying to look at neural progenitor populations along the lateral ventricles so tearing there is no good. Brains were fixed in 4% PFA overnight then put in 30% sucrose for ~72 hrs until they sunk. Tissue was embedded in OCT and put in dry ice for ~20 minutes to freeze. Sections were cut at 8um in a cryostat where chamber temp was -20C and stage temp was -17C. Any tips on how to avoid these tears would be much appreciated.
These are some possible reason for this
Sample not being fixed long enough. Not completely frozen, or cold enough.
Possible Solutions
Be sure to note that the sample, even if frozen, can warm up causing issues.
So temperature control is paramount.
1: Drain all access solution from the fixed tissue by wicking with a tissue or filter paper, then place the tissue into the embedding media in a plastic mold and fast freeze (use isopentane cooled in liquid N2 and not directly in liquid N2 is because the emdebbing media and/or the tissue itself can crack when N2 becomes gaseous and bubbles.) it, then put it in the -80 freezer in a sealed zip-lock bag for at least a few hours before cryo-sectioning it.
2: Make sure the OTC is fully frozen before sectioning. (if the boundary of OCT and tissue is gray and is not white suggesting that the OCT is not cold enough near the tissue)
3: Also if the OCT surrounding the tissue is thin around the edges. This may causes compression and you may see cut in your tissues (the tissue is not supported enough), which may hack off or tear your samples.
4: Rolling is normally solved by proper alignment of the roll guard to the blade, changing the cutting speed, or by changing the blade angle.
So check whether its your anti roll guard that is damaging the tissue as it slides through.
Aamir Sharif thanks for the tips! Seems that there was too much excess sucrose still in the brain during the freezing process. Wicking the excess sucrose off with a kimwipe and let it sit out on a kimwipe for ~2 min let to much better sections. Not completely free of tears but significantly better.