Question: How do I fresh freeze and embed my tissue in OCT?
Answer: There are two options to fresh freeze and embed your tissue in OCT in preparation for running Visium.
- Snap freeze the tissue and store it in a sealed container at -80C. Embed tissue in OCT before cryosectioning.
- Prepare an isopentane and liquid nitrogen bath. Place tissue using forceps or spatula into isopentane until completely frozen ~1 min depending upon tissue size. Transfer tissue into pre-chilled container on dry-ice. Transport container on dry ice to -80C or liquid nitrogen for long term storage. Failure to use a sealed container for storage may lead to tissue damage due to dehydration.
- Simultaneously freeze and embed tissues in OCT and store in a sealed container at -80C until ready to section.
- Prepare an isopentane and liquid nitrogen bath. In a petri dish, carefully coat fresh tissue sample with room temperature OCT. Using a spatula, place the OCT-coated tissue into an appropriately sized cryomold. Cover the tissue completely with OCT. Label the cryomold to mark the orientation of the tissue. Using forceps, lower the embedded tissue into the isopentane without fully submerging. Keep cryomold in contact with isopentane until the OCT has solidified and turned white. Once frozen, place the cryomold on powdered dry ice. Store frozen embedded tissue in a sealed container at -80C or liquid nitrogen for long-term storage.