I work with fish muscle pieces of about 4x2x3 mm3 and I will need to cut at 10um.
I have prepared some blocks of tissue following the standard procedure: cooled isopentane in liquid nitrogen to freeze non-fixed fresh muscle tissue, previously embedded in OCT. I have done the same with dry ice instead of liquid nitrogen with the same results. I’ve made 10 to 14um and the result was that the majority of the muscle fibres appeared to be broken (there is only the membrane!) and there were small bubbles inside the tissue.
I have seen several posts of muscle cryosectioning following this freezing method, so I don’t know if I have missed an important point during the freezing procedure, i.e. how do you know the isopentane is ready? do you recommend to embed first in sucrose? what about the storage of the blocks?...
Does anybody know what would be the reason?
Thank you all in advance!
Have you tried to cryo preserve following fixation before you embed in OCT?
following fixation (IN ? 4% PFA) blot excess fixative with Whatman filter paper and transfer into 4 vols of 30% sucrose made up fresh or stored at -20 (as long as it is sterile) and if FIXED leave at room temperature. (If unfixed leave at 4 degrees)
Leave in sucrose until the tissue has sunk to the bottom (depending on size of tissue usually takes from 25 minutes to hour). Remove and blot excess sucrose using Whatman filter paper. Excess sucrose will freeze but when sectioning, has a different melting temperature to OCT and will cause you problems if not removed. Transfer to OCT and freeze as you have been doing. Store at MINUS 20 NOT minus 80 ! OCT does odd things at this temperature.
Thank you very much Caroline, I really appreciate it! I'll definetely follow your recomendations. I did not know that is better to keep the blocks at -20 rather than -80. Is this only recommended when using sucrose? Is it recommended also for long time storage of the blocks?
Cheers!
No... if blocks are required for cryosectioning and are in OCT then they should be stored at minus 20. OCT turns into stringy gloopy stuff when transferred from minus 80 to warmer minus 20. Also your tissue even if cryoprotected will look shocking ! with alot of debris
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