[Histonet] Re: Removing frozen tissue blocks from metal disks after cryotomy

[gayle callis]

Andrea, 

 

We seal a cryosectioned block with a very thin layer of OCT first.  How to's
follow: 

 

Use a blade guard, lock flywheel and put a drop of OCT on your thumb, rub
quickly across block face and make sure tissue is "smeared over" with a thin
layer of OCT.  We want this layer to freeze as quickly as possible.   We do
this while the disk is still in the microtome holder, and can then mark
(with a sharpie marker) the block if you need to identify.  Don't put a huge
blob of OCT on the face as this warms the tissue too much until it freezes
again. 

 

Take the disk out of holder, and warm the back of disk with hand.  Our disks
have stems so placing the stems between the fingers warms the metal allows
warming the back efficiently,  then the OCT warms but does not melt the
block.  The block should pop off the disk by using a thin, metal spatula,
dull but thin blade paring knife, or a mini-cheese spreader recycled from a
kitchen - the small ones used at parties for spreading cheeses, etc or a
single edge razor blade inserted NEXT to the disk, between disk and OCT,
then twisting.  We use single edge razor blades(teflon coated)  at RT, so
this works even better.   It is better to use a thin, dull metal edge for
this instead of razor blades to prevent bad cuts to your fingers.   We have
even lined up sealed blocks in a plastic micro-centrifuge tube block holder
- at room temperature,  and let the OCT start to warm up for block removal.
This requires careful attention so the blocks do not start to melt, so work
quickly.  Some people are sensitive to cold, and this is another way to do
this or keep changing the block around to be between different fingers.  

 

Whatever you do, DO NOT hold the disk up and out of chamber and then try to
cut the block OFF the disk.  Do NOT try to cut through the OCT if the back
of block has not been warmed.  I have had people cut themselves with razor
blades trying to do this.  If you brace the disk in the little brush holding
tray in front of knife holder , you can steady the disk/block, and pop the
block off without cutting yourself.  Sort of like cutting a slab of cheese
or salami.  

 

After the block is removed, we trim excess OCT from block where the media
has spread out during  freezing onto the disk, in other words, reshape the
block back to a square or rectangle.  This is so the block fits nicely into
storage tube. Wrap the whole block with aluminum foil, and slip the blocks
into 50 ml centrifuge tubes.  The foil can be marked with sharpie marker to
identify the tissue.  We keep aluminum foil squares on hand for this
purpose. 

 

We keep cryosectioned tissue blocks for years, and have had total success
doing murine CD markers even after 7 years of storage.   The key is to
reseal the block, and store at  -80C. 

 

Gayle M. Callis

HTL(ASCP)HT,MT

Bozeman MT