A Frozen embedding unfrozen tissue in OCT compound - michelletytong/TRGProtocols GitHub Wiki

Materials & Equipment

  • OCT
  • aluminum foil
  • permanent maker
  • forceps (small) - for manipulating samples
  • forceps (large) - for holding samples in liquid nitrogen
  • liquid nitrogen (or dry ice)
  • ziplock bag
  • needles

From tissue

  1. Label the sample ID on the surface of the Cryomold (made from aluminum foil) with a Permanent Maker.
  2. Place few drops of OCT (depends on the size of the tissue to be embedded) onto the center of the bottom of cryomold. Be careful to select the proper size embedding mold according to the size of the tissues to be embedded.
  3. Place the unfrozen tissue sample in the drops and oriented. Make sure that the side touching the bottom of the cryomold is the side you want sectioned first. Gently push the tissue with a forceps to ensure that the bottom surface of the tissue is placed properly, level with the container, touching the face of the bottom and the tissue is located in the center of the mold. Be very careful to orient the sample because it is important for the demonstration of proper morphology.
  4. Carefully drop more OCT onto the specimen until it is completely covered. None of the tissue should remain exposed.
  5. Try to avoid the formation of air bubbles. Remove any bubbles inside the OCT by carefully popping them with a sharp needle, e.g. This is important because the air bubbles will create problems when cutting sections.
  6. Let it settle for 15-30 seconds to allow the OCT to completely wet the surface of the tissue.
  7. Place cryomold with OCT covered sample in it on the surface of the cold isopentane/2-methylbutane or in the vapor phase right next to the liquid nitrogen with the flat side down using a long forceps.
  8. After hardening of the OCT compound (it will happen in 0.5-1 minute), wrap the OCT embedded block in foil and placed in a labeled zip bag.

Storage

The frozen blocks can be temporary stored in dry ice. Transfer the blocks to a liquid nitrogen storage tank (Years) or -80°C freezer (Months). From this point over, the sample should never be thawed unless there is specific requirement.