Freezing Tissue For Cryostat Sectioning

All tissue types except muscle:

  1. Tissue should preferably be snap-frozen by quenching in liquid nitrogen.
  2. Quenching is accomplished by putting a small amount of OCT compound onto a cork disc, placing the freshly biopsied tissue onto that and then dropping the disc upside down into the liquid nitrogen.
  3. Upon reaching the laboratory the frozen sample is attached to a cryostat chuck. To achieve this put a small amount of OCT compound or tragacanth gum onto a chuck. Quickly remove the sample on the cork disc from the liquid nitrogen, place it on top of the tragacanth gum and re-immerse the whole in the liquid nitrogen with a pair of long handled forceps until frozen.
  4. The sample is then put into the cryostat where it must be left to warm to cryostat temperature before cutting, 15-20 minutes is usually adequate. However, urgent specimens from the operating theatre need to be cut as soon as possible so sectioning should be attempted immediately.
  5. If the sample reaches the lab without the benefit of liquid nitrogen quenching it may be frozen using CO2 apparatus.
  6. If the sample has been fixed it must be washed in running water for several hours to remove the fixative or it will not freeze properly and cutting will be very difficult.

Use of the CO2 freezing apparatus:

  1. Make sure the CO2 cylinder tap is turned OFF and that the tap on the freezing apparatus is OPEN (anti-clockwise).
  2. Place a small amount of OCT compound or tragacanth gum onto a chuck.Place the tissue sample onto this and attach the chuck to the freezing apparatus.Make sure the chuck is held firmly by tightening the screws.
  3. Attach the safety cover.
  4. Turn the tap on the freezing apparatus clockwise to CLOSE it.
  5. Turn ON the CO2 cylinder tap.
  6. Hold down the safety lid with one hand whilst turning ON the tapon the freezing apparatus (anti-clockwise) to release CO2gas. Use short bursts until the sample is completely frozen.
  7. CLOSE the tap on the freezing apparatus (clockwise).
  8. Turn OFF the CO2 cylinder tap.
  9. OPEN the tap on the freezing apparatus (anti-clockwise) to release any remaining gas.
  10. Remove the safety lid, release the chuck and place it in the cryostat.Allow to equilibrate for 10 - 15 minutes before cutting.

Frozen Sections From Muscle Biopsies :

Muscle is the exception when freezing tissue for cryotomy. It must be frozen using cold iso-pentane (2-methylbutane) or ice crystal artefact will occur. (This should include other tissue containing muscle such as full-depth gut samples.)

  1. Take unfixed muscle and cut to an appropriate size and orientation.
  2. Place the tissue on a spot of OCT on a cork disc so that you willcut T/S (transverse section).
  3. Put a small amount of liquid nitrogen in a thermos flask - about 5cm deep.
  4. Place a cold-resistant plastic beaker containing some iso-pentane into the liquid nitrogen.
  5. Wait until the iso-pentane is syrupy and almost solidifying. If it does solidify take it out of the nitrogen and stir it with a plastic spatula until it is mostly liquid again.
  6. Plunge the cork discs, tissue side down, into the iso-pentane. This can be done out of the thermos flask.
  7. Attach the cork disc to a cryostat chuck as described above.
  8. Put the chuck into the cryostat and leave to reach temperature for 15 - 20 minutes.

Storing Frozen Tissue Samples For Extended Periods:

Sometimes small tissue samples needs to be kept frozen in storage for later investigation. In order to prevent deterioration of the samples and embedding medium due to dessication they should be stored in well sealed bags or containers. In this laboratory it has been found that samples frozen in OCT onto cork discs, wrapped in foil, placed in a plastic bag and then sealed in a zip-lock bag will keep well at minus 70oC for at least 2-3 years and maybe longer. However, if the sample is kept in a container which is not air-tight it will eventually freeze-dry. The OCT then becomes plastic-like and is almost impossible to cut.

Using The Slee Cryostat :

Temperature gauge and light switch.
Micrometer and anti-roll plate control
Main cutting handle and advance mechanism handle.
OCT compound, cork disc and chuck
Internal view of advance mechanism and anti-roll plate.