HISTOTECHNOLOGY AND COMPARATIVE PATHOLOGY FACILITY

Frozen Sections

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.
       
 
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