Recently I snap-froze some tissue sections to slice them using the cryostat, but when I took the blocks out of the -80 one of them didn't freeze properly, and was soft and fell apart when I tried to slice it.
I remember reading once that OCT may not be freezing properly (I don't recall why). Has someone experienced something like this before and how did you avoid it?
How do you prepare or pretreat the samples before freezing? What snap-freezing method is yours?
Look like Sebastian and Shyamala both have a problem with too soft blocks once in a while. POssibly this is due to substances that lower the freezing point of the tissue. Or there is too much fat in the tissue. How do both of you prepare the biopsies prior to snap freezing them. What it the temperature of the cryostat? What tissue are you having problem with and is there a type of tissue that doesn't give trouble?
The best way to freeze samples in OCT is using liquid Nitrogen. Make sure your sample is covered in OCT (with no air bubbles in it). Float the plastic mold on the surface of the liquid Nitrogen (don't just trow it in). Take it out while there is still liquid/clear OCT in the middle of the block. Leave it on the counter and watch the OCT continue to freeze and the liquid area closes. It is takes too long, immerse the block in the liquid N for a little longer. Don't let it sit in the liquid N!!! It will over cool and crack!
Keep blocks at -80. When ready to section, place in the cryostat so the temperature increases gradually to the correct temperature for your tissue - about 15 - 20 min. Never try to cut tissue that is still -80.
Most frozen sectioning is done between -13 to -20 degree. Requirements for different tissues vary. If your sections crumble, increase the temp. If the section doesn't form, but looks like crinkled tiny deposit at the edge of the knife, you need to cool your block surface by spraying as Shyamala recommended or lower the temp in the cryostat.
Good luck with it
I have never encountered this problem, but I always leave the mold With Tissue-tek and the sample in -80oC at least over night. In addition I section it somewhere between -15 and -20oC.
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