[Histonet] Thawing frozen tissue

[Mesru T]

Hi Maria,

Often people freeze the tissues right after dissecting them out of the
animal because either they are not sure what to do or they do not know
better.
Then they bring the frozen tissues kept at -80 C freezer  and request
paraffin embedding.
I usually prefer to thaw them gradually. I place the frozen tissues in the
cryostat (in a tightly sealed zip lock bag),  which is at -50 C. Then I
increase the temperature of the cryostat by 5 C every 45 min minutes until
it is -5 C. Then I prepare ice cold 10% NBF (or 4% PFA)  and place the
tissues in the ice cold fixative and let the tissues fix at room
temperature for enough time depending on the size and the type of tissue
(For my tissues I fixed overnight at room temp). After that I process for
paraffin embedding. The H&E staining comes fine for frozen tissue. And I
have tried IHC with 4 different antibodies and had excellent results. I
have tried mouse brain tumor ( pieces like ~1cm3 in volume) , human brain
tumor ( small pieces like~ 0.5cm3 in volume) and human lymph nodes (pieces
like ~3mm3 in volume) with good results. You can also re-freeze after
thawing but you may have poor tissue architecture.
I would suggest you to do a pilot trial experiment. You can thaw one block
that you can spare (or you can prepare a new samples just to test thawing)
 and test how it comes out for your particular case. If you have
satisfactory results you can go ahead and thaw other samples. I have tried
to section frozen gelatin embedded tissue in the past and it was very
difficult. Please take images with your phone and send us the photos of the
defective blocks.

Good luck!
Mesru