Frozen services

Tissue should be frozen as soon as possible after removal from the animal to preserve tissue morphology. Tissue can either be un-fixed (fresh-frozen) or fixed (fixed-frozen) prior to freezing.

There are several different methods to freeze tissue. The Core facility freezes tissue in a container with isopentane that has been chilled with dry ice.

Tissue is fixed in either 4% paraformaldehyde (PFA) or 10% Formalin as soon as possible after the tissue is removed from the animal. The fixation time is dependent on tissue sample size, usually 24 – 48 hours. The tissue is rinsed in PBS and then transferred to a 20% or 30% sucrose solution in PBS. The sucrose solution is then replaced three times (first and second change after one hour, third change after 2 hours or leave overnight). Samples are now ready to bring to the Core.

Sucrose acts as a cryoprotectant and is highly recommended to use after fixation to preserve tissue morphology. It prevents ice crystal formation in tissues when water freezes and expands. Ice crystals can break cell membranes and produce holes within cells creating “Swiss Cheese” artifacts. Other cryoprotectants that have been used include glycerol and polyethylene glycol.

Usually OCT (Optimal Cutting Temperature) compound is used to embed frozen tissue. This is done during the freezing process in a cryomold. OCT can also be added to the chuck prior to sectioning to provide support to the tissue.

Frozen sections are quicker to prepare than paraffin and are more suitable for molecular analysis (DNA/RNA studies, enzyme studies and post-translational modifications) since the proteins are not denatured as they are in paraffin. They are usually thicker (8 um or >), provide lower resolution than paraffin sectioning and less stable (must be kept frozen). The Core facility has well established protocols for routine/special stains and IHC/IF staining on frozen sections.