Laboratory Animal Sciences Program
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Freezing Procedure for Histopathology Specimens

Tissues that are not properly frozen will suffer severe disruption of tissue architecture due to the formation of ice crystals. Once ice crystals have formed, there is no remedial action that will restore the tissue, therefore the following procedure is recommended for submittal of tissues for cryotomy:

Materials

  • Freezing mixture: prepare a “slush” with dry ice in isopentane (more efficient than dry ice alone)
  • OCT molds (supplier- Tissue -Tek® Cryo Molds #6557)
  • OCT (supplier- Tissue -Tek® #4583 (25820 5mm))
  • Cryo-spray (supplier- Surgipath “Frost Bite” #03100)

Fresh Specimens

  • Fresh tissue should be frozen as quickly as possible
  • Trim the tissue to a thickness of 3-4mm
  • Whenever possible embed one piece of tissue per block (mixing tissue types-e.g. liver and skin- in the same block should be avoided)
  • Place the tissue (cut side down), into an “OCT” mold and surround with OCT.
  • Place the mold onto the freezing mixture
  • Spray the upper surface with cryo spray
  • Keep the tissue on the freezing mixture until all of the OCT turns white; then allow it to set for an additional ten minutes
  • Note: OCT freezes at a different rate to tissues, frozen OCT does not mean that the tissue is frozen. Fatty tissues need more time to freeze.

    Note: Once tissues are frozen, maintain them at between –20 °C and –80 °C DO NOT ALLOW TO THAW!

Fixed Tissue

Fixed tissues should be sent directly to PHL for freezing. If it is necessary to freeze fixed tissue please contact PHL for advice.
Larry Sternberg, Histology Manager, PHL
Phone: 301-846-6881, E-mail: sternberglr@mail.nih.gov

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