I currently have a problem. When I immerse skin tissue in O.C.T. and freeze it in liquid nitrogen the cryosections come out great (uninfected skin control) but when I do this exact procedure with my tissue of interest (infected skin) and start cutting, it seems to have no matrix in which to “hold on to” and falls apart after sectioning. I think I need to fix this tissue previous to cryocutting.
Did you leave the tissue in OCT for a period of time before freezing it? Sometimes when you freeze the sample before water equilibration is done, there will be shards of OCT around the sample. If the sample is pre-fixed, you can keep it in OCT (preferably inside the mold of it's own) @ 4C frig overnight then snap-freeze it.
Another possible cause is the carryover of small air bubbles around your sample. The OCT is very thick and sticky, which tend to lock air bubbles in it. After immersing the sample in OCT, take a closer look and remove the bubbles with micropipette.
Good luck!
Dear Angel Francisco,
It is always need to fix your samples. Samples fixation will avoid autolysis, thus preserving your samples. However, it in cryo-embedding, you can both fix your samples before or after sectioning. Considering the problem you comment, I would fix the samples before embedding. Moreover, it is recommended to cryoprotect your samples before freezing. I then suggest you this protocol:
-Fix your sample in 4% PFA (few hours to o/n - depending on the samples thickness).
-Rinse 3x10 min with PBS.
-Leave your sample in 15% sucrose in PBS at 4 ºC, until tissue sinks.
-Transfer your sample to 30% sucrose in PBS at 4 ºC, until tissue sinks.
-Remove the liquid excess around the tissue (with a paper-towl, or similar).
-Continue with the OCT embedding and freezing protocol.
Good luck!
Thank you Yi-Chun and Alex for your time, advice and shared knowledge, I will put your methods to the test and get back to you.
Thanks again!
I agree with both Yi-Chun and Alex. However, you could directly, without any pre-treatment, preserved the tissue samples directly in OCT in the following manner. Fill half way though the tissue holding cassette with OCT, place the tissue sample on top of it and add more OCT till the sample is completely submerged. Wrap the cassette with an aluminum foils, float it carefully on liquid nitrogen till it sinks. Finally, transfer it into a cryopreserve storage tank or - 80 degree Celsius freezer for storage.
Hi Angel. Sorry to hear your problem late.
Tissues, in particular skin, become bristle and ruptures at sectioning after they have been snap-frozen in liquid nitrogen (-196oC). Emerge your tissue with OCT in a cassette in an orientation you to cut it later. Fill 1/4 a beaker with isopentane (melting point ~-160oC), lower it into a thermos containing liquid nitrogen a couple of minute till isopentane looks opaque (reaching ~-160oC). Emerge the cassette containing tissue/OCT into freezing isopentane for a few tens seconds (till frozen white).
Some people also through dry ice pellets into isopentane to get it cold but I guess the temperature would be around -60oC the lowest by dry ice.
Other people including me during my PhD soak tissues in sucrose before OCT embedding to get them soft at sectioning. In my experience it leads to fault positivity on TUNEL staining but otherwise is fine for immunostaining.
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