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 70^oC 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.

• Open the main window and remove the insulating block.

• Switch on the internal light, the switch is down near the temperature gauge.

• Check that the temperature is at -20^oC which is the standardoperating temperature.  If you are cutting soft tissue such as liver you may need to raise the temperature to ^-15^oC  and any specimen that has been formalin fixed is probably best cut at ^-10^oC. Harder tissues produce better sections at lower temperatures and vice versa.

• Fixed tissue MUST be washed in running tap water before freezing. See above.

• Once the tissue is attached to the chuck it should be left in the cryostat for an adequate length of time to allow it to reach the selected operating temperature. See above

• Check that the cryostat advance mechanism is at the top of its travelon the central thread.  If it isn't rotate the main cutting handle so that the chuck holder is at the lowest point of travel and then wind the small handle on the top of the cryostat in a clockwise direction.  Keep doing this and watch the advance mechanism arm travel up the central thread until it almost reaches the top, don't go too far or it may stick.

• Attach the chuck to the cryostat arm and tighten both wing nuts to hold it firmly.  Try to align the tissue so that it's flattest side is parallel to the knife edge.  This will help to achieve even cuttingof the block and prevent sections from crumpling.

• If the knife is too far forward or too far back you can move the assembly by releasing the handle on the right of the knife holder; push it away from you.  Slide it to the desired position and tighten the handle again.

• Adjust the section thickness using the micrometer screw on the lefthand side of the cryostat.  Most tissues cut well at 6-8µ but you may have to increase this for difficult tissues.

• If the anti-roll plate is in position rotate it out of the way usingthe small handle on the left-hand side of the cryostat.

• Trim the tissue block so that you have a complete section through the tissue.  This can be achieved by simply winding the main cutting handle (takes a long time) or by winding the handle whilst also winding the small handle anti-clockwise on top of the cryostat.  This can be a little tricky at first but is the quickest way of trimming a block.

• Remove all traces of tissue and ice from the knife edge by brushing firmly UPWARDS with a stiff bristle paint brush.

• Use the handle on the left of the cryostat to bring the anti-roll plate back into position.  It should be exactly parallel to the knife edge and when looking at it through the small circular window it should also line up with the knife edge.  failure to cut sections is most often caused by a mis-aligned anti-roll plate.

• Turn the main cutting handle to produce a section. The speed at which you turn the handle and cut the section depends on the type of tissue you are cutting.  Generally the harder the tissue the faster you need to cut it.  Usually more than one section or even a ribbon of sections are cut for each slide.  This increases the chances of having at least one good section per slide.

• To pick up the sections have a slide ready.  Use plain slides for unfixed tissue and slides coated with gelatine or poly-l-lysine for fixed tissue.  Rotate the anti-roll plate out of the way.  Hold the slide between your fore finger and thumb, open the small window and place the slide end against the left hand knife clamp and swivel the slide down onto the sections and hold it there for a few seconds to allow the sections to melt onto the slide.

• Remove the slide and allow it to dry at room temperature for several minutes before you attempt any staining.

• Again brush off all remaining tissue and ice from the knife edge making sure you brush in an upwards direction.

• Repeat as necessary.  When you are done remove all tissue wastefrom the machine and reset the advance mechanism to the top of its travelfor the next user.

• Replace the insulating block and switch off the internal light.