Frozen section histology requires the use of fixed or un-fixed tissue samples
- If tissue is not being preserved by a fixative, it is imperative that the tissue is frozen as quickly as possible after removal from the animal
- Freeze in isopentane chilled to at least -80 in dry ice
Tissue samples should be small (3mm x 3mm) to avoid slow freezing of the sample, which can result in artifacts
- Dab tissue on paper towel to dry to reduce crystal formation during freezing
- Place tissue into mold and surround with OCT
- Remove bubbles
- Place mold in isopentane with flat face down, when frozen, drop mold into chilled isopentane
- If not pre-fixed, fix sections in cold 100% methanol or acetone for 2-3 minutes and store at –80 or –20C
Frozen tissues with special needs
- Frozen brains—how to get rid of bubble artifact
- Best to fix brains by immersion or perfusion followed by immersion lPlace brains in a 30% sucrose solution in PBS O/N (no longer) lEmbed in OCT and freeze as described
- Frozen muscle
- Prepare 2-methylbutane chilled in liquid nitrogen (2-methylbutane must be very cold such that it is slightly frozen on the bottom of the container before you freeze the muscle)
- Don’t pull or break muscle during dissection
- Using a small drop of OCT, adhere one end of the muscle to cork or cardboard
- Turn upside down and immerse in 2-methylbutane for 15-25 seconds and then place on dry ice (if you leave it too long in this super-chilled reagent, the sample will crack)
- Store at -80
- NEVER cover it with OCT! These must be frozen very quickly to avoid severe artifacts. Don't freeze them in liquid nitrogen alone, either.