Fixation and Shipment of Tissue Samples for Frozen Downstream Applications

The idea of fixing and shipping samples for frozen sectioning can seem like an intimidating task. In reality, it’s much more straightforward than preparing tissue for paraffin applications. Before you start collecting your tissue samples, here are some tips and considerations to optimize your results for OCT services.

Fresh Frozen and Fixed Frozen
There are two methods of tissue preservation after collection, fresh frozen and fixed frozen. Fresh frozen requires isopentane and liquid nitrogen to immediately freeze the tissue after necropsy while fixed frozen follows the traditional procedures of fixation with a fixative.

What is the Right Fixative for Fixed Frozen
The downstream applications of your study will determine the right fixative to use during fixation. For most frozen applications with immunohistochemistry (IHC) and immunofluorescence (IF), 10% neutral buffered formalin (NBF) or 4% paraformaldehyde (PFA) are frequently used. Methanol and acetone can also be used as fixatives that preserve lipids and cellular membranes for immunohistochemistry (IHC) and immunofluorescence (IF) assays.

Preparing Tissue Samples
Once you have determined the right fixative, samples can be fixed through perfusion and/or immersion fixation. Perfusion is a method of preservation that injects fixative solution into the bloodstream by cardiac or vena cava puncture. Immersion involves immersing the tissue in the fixative. To ensure proper fixation, the sample should be immersed in the appropriate fixative with a minimum volume of 20 times the sample volume. It’s also important to consider fixation time - over fixation can cause tissue hardening while under fixation can result in poor morphology preservation. For most studies, fixation times of 24-48 hours maximum at 4°C are preferred for preservation.
If you plan to send tissue samples fresh, immerse the tissue in an isopentane bath chilled with liquid nitrogen for at least 1 minute or until completely frozen. Once frozen, transfer the tissue to a pre-chilled tissue embedding plastic mold on dry ice and embed using pre-chilled OCT at 4°C. An ideal snap freeze should freeze down samples quickly and evenly to reduce ice crystal formation. However, freezing too quickly can also cause tissue to crack.
To prevent freeze damage and ice crystal formation, a cryoprotectant solution such as sucrose in TBS can be used to cryoprotect fixed tissue samples. To cryoprotect the tissue samples, immerse the tissue in 15% sucrose overnight at 4℃. Once the tissue sinks down, repeat the process using 30% sucrose in TBS solution.

Storage and Shipping
Once the samples have been snap frozen or fixed and cryoprotected, they can be shipped to us, either with plenty of dry ice for fresh frozen samples or at 4°C (ice packs) for fixed samples. If you plan to store tissue samples for long periods of time before shipping them to us, consider embedding the samples immediately in OCT and storing them in a -80°C freezer. From this point, the sample should never be thawed unless necessary.
When preparing samples for shipment, the temperature conditions and the shipping method must all be carefully considered. Shipments should be made earlier in the week to avoid delays into the weekend. Other considerations such as proper sample labeling, documentation, packaging, and cushioning can help ensure that samples arrive in good condition and on time for analysis.
For more suggestions or advice on how to best collect and fix your tissue samples for frozen downstream applications, contact info@waxitinc.com or read our frozen fixation protocol for more in-depth details.