[Histonet] Re: Baboon Tissues

[Johnson, Teri]

Mike,

It sounds like a classic case of either slow freezing or thawing and slow re-freezing causing ice crystal formation. One other guess is that they were fixed in formalin but not cryoprotected, and then frozen. I have seen freezing artifact in this way as well. Unfortunately there is nothing you can do at this point to save the samples. They are architecturally ruined.

It is entirely possible to snap freeze unfixed tissues properly without a sucrose gradient. Matter of fact, we only use the sucrose cryoprotection step on samples that have been previously fixed. As for the proper collection of the new samples, that depends on what will need to happen to them after they are mounted on the slide. The best histology results from fixed and cryoprotected frozen section. You can usually get something that looks almost as good as a paraffin section. Arguably the best IHC is achieved on unfixed and snap frozen tissues. Gayle Callis is the master at doing this, she has worked in rodent spleen in cryo for many years and can give you tons of good advice on sectioning and staining them. You should do a histonet search looking for her information on sample handling if you indeed need unfixed frozen sections.

For fixed frozens, definitely put them through the sucrose gradient and then snap freeze. Make sure your sectioning temperature is not too cold (shattering of the tissue), or too warm (ooey gooey sticky mess). You are looking for the Goldilocks temp - just right.

Good luck!

Teri Johnson, HT(ASCP)QIHC
Head, Histology and Electron Microscopy
Stowers Institute for Medical Research
Kansas City, MO