Download - Preparation of Frozen Tissues for Sectioning
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8/6/2019 Preparation of Frozen Tissues for Sectioning
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Preparation of Frozen Tissues for Sectioning
Materials
O.C.T. compound
Cryomold (25mm x 20mm x 5mm)
Dewar or ice bucket to hold liquid nitrogen.
Isopentane (2-Methylbutane)
Small container to hold isopentane
Long handled forceps
ring stand and clamp
Ice bucket for dry ice
Procedure
1. Fill dewar with liquid nitrogen.2. Fill small container with approximately 2 inches of isopentane.
3. Suspend isopentane container from clamp on ring stand and lower the
container into the liquid nitrogen. The base of the container should just touch
the top of the liquid nitrogen and cause it to bubble as it cools the container. If
you insert the container too far into the liquid nitrogen the isopentane will
freeze. If this happens, just remove the container and allow the isopentane to
thaw and then place it back in the liquid nitrogen.
4. Label a cryomold with the identifying data of the tissue. Make sure to use a
marker resistant to organic solvents!
5. Place a small amount of O.C.T. in the bottom of the cryomold.
6. Remove the tissue to be frozen.7. Place the tissue oriented with the side you are interested in at the bottom of the
cryomold.
8. Fill the cryomold with O.C.T. compound. Use caution not to move the tissue
when adding the O.C.T. and be sure to remove any bubbles that form near the
tissue with a forceps as these will cause problems with sectioning (cracking
and fragmentation).
9. Place the cryomold into the isopentane. Use caution not to tip the cryomold
and dislogding the tissue. A little preparation here and ordering a pair of long
handled forceps that can open wide enough to grasp the entire cryomold help
here quite a bit.
10. The O.C.T. will freeze into a solid white color. The tissue should be coveredand not visible as you look down on the mold. Allow approximately 1 minute
for freezing of the entire block.
11. Transfer the tissue to and ice bucket with dry ice and continue with the rest of
the tissues.
12. Place tissues in plastic bag or other airtight container and store at -80oC.
Notes: For some tissues (particularly lung and intestine) it is advantageous to infuse
them with a solution of 50% O.C.T. and 50% PBS prior to placement in the cryomold
to preserve the tissue structure.