FREEZING TISSUE FOR CRYOSTAT SECTIONING

FROZEN SECTIONS FROM MUSCLE BIOPSIES:

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

USING THE SLEE CRYOSTAT:

• 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^o C which is the standardoperating temperature.  If you are cutting soft tissue such as liver you may need to raise the temperature to ^- 15^o C  and any specimen that has been formalin fixed is probably best cut at ^- 10^o C. 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.