Protocol for 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 covered and 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 -70^oC

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.