Tissue Fixation and Freezing Protocol

1.) If possible, perfuse tissue in situ with 1X PBS followed by 2% paraformaldehyde made in PBS (directions available at CBI). Place tissue in 2% paraformaldehyde for and additional 1-2 hours. The tissue needs to be completely submerged in the solution. Larger tissues need 2 hours incubation in 2% paraformaldehyde in PBS (lobe of liver). One hour is sufficient for a small tissue such as mouse lymph node or hollow organs like rodent intestines.

2.) Place tissue in 30% sucrose for 24 hours having changed the sucrose 2-3x within 24 hours. Provide enough 30% sucrose to submerge the tissue.†

3.) Place a plastic beaker containing 2-Methylbutane (Fisher Cat No. O3551-4) in an ice bucket, Styrofoam box or equivalent container containing a few inches of liquid nitrogen. Allow the beaker to cool for several minutes. Beaker with 2-methylbutane is to remain in the ice bucket submerged in the liquid nitrogen during the entire freezing protocol.

4.) Samples, fixed as above or fresh, can be placed on small pieces of filter paper manipulated with forceps (the tissue often freezes to the forceps if picked up directly). Alternatively, the tissue sample can be embedded in OCT compound in a small cassette.

5.) Pick up sample and completely immerse in the liquid nitrogen cooled 2-Methylbutane for 30 seconds.

6.) Remove sample from beaker and immerse in the liquid nitrogen for an additional 10 seconds.

7.) Immerse a properly labeled, suitable box or cryopreservation bag in the liquid nitrogen for a few seconds to cool container to the same temperature as the tissue.

8.) Place sample into the storage container/bag and immerse in the liquid nitrogen until placing sample into -80oC storage.

9.) Samples to be transferred between labs or waiting sectioning should be stored on dry ice to avoid thawing.