I am trying to do Laser Capture Micro-dissection on samples that have been frozen at -80C, for many years. My purpose is to isolate specific cell type and isolate RNA for RNA-seq experiment. I have read some reports saying that it is not possible to use OCT if your tissue is already frozen. My question now if this is just a technical issue that can be solved or some serious problem with no solution?
Dear Waleed
I have been working with post mortem brain tissu and laser microdissection. I did what you suggest (embedding samples already frozen with OCT medium); you can try.
As I remember, you will have a difference of texture between your sample and the OCT medium which surrounds your sample.
The OCT will allow your sample to stick on the plot in your cryostat.
What may happen is that your sample will not be attached enough with the OCT. In that context, when your sample touch the blade of the cryostat, instead of getting a slice, the sample might be detached. To avoid this, I put OCT below and around the sample then keep the preparation in the cryostat for 5 minutes in order to get the OCT well frozen in contact with the sample.
Something else which may help is to leave the sample of interest from the -80°c freezer and put it into the cryostat (-20°C) 15 to 20 mins before work. Consequently, the difference of temperature between the sample and the OCT at the time of preparation would be less important.
Sincerely
Gilles
Thank you very much Gilles. That is a very good point to start with.
Hi Waleed,
I have done laser microdissection on frozen tissues (not many years though).
I faced no problems. Few things which I considered while doing cryo sectioning are:-
for sectioning, I embedded the frozen tissue samples in OCT. I used to cover the samples by OCT on all sides.Then leave the sample in cryostat for some time as suggested by Gilles.
The cryostat I used had an option for fast freezing. Once you fix your sample on the holder of cryostat, this option quickly lowers the temperature (approx. till -35°C) of the holder as compared to chamber temperature. I used to keep the samples for 15-20 minutes on fast freezing and then shift to normal mode.
If you have such option in the cryostat, try using it, its helpful.
Best wishes
Sushma
Hi Sushma,
Thank you very much for the advices. I have actually started doing some trails now and so far have no problem. I am doing what you both mentioned by keeping the sample for like 20 minutes to equilibrate the temperature before starting the sectioning.
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