Cryostat-Microtome Sectioning

The lab is equipped with a sliding microtome,and two cryostat microtomes. The Leica cryostat microtome is the true work horse of the lab. Virtually all sectioning of both fresh frozen and perfused tissue is done on this machine. This document only covers sectioning on the Leica Cryostat microtome.  This article presumes that the tissue is either fixed in formalin  or has been  frozen prior to sectioning.  The cryostat  has a quick freeze feature which allows a fresh brain to be  frozen directly in the cryostat, but this procedure will not be covered in this web page. 

Procedure

• Roll back the back rest for the knife as far as possible using the knurled dials on each side of the knife holder.
• Carefully place a sharp knife into the knife holder (note that the D-profile knives must be inserted into the cryostat with the handle hole to your left. The C-profile knives can be inserted either way).  Be careful to avoid touching the knife edge against the guards or any other surfaces. Advance the knife back rest using the knurled knobs until the knife edge is about 1-2mm away from the end and secure in place. Allow sufficient time for the knife to cool.
• Using the coarse feed on  control panel 2 move the specimen holder to the rear of the chamber. Lock the handwheel. (The handwheel can only be locked when the handle on the handwheel is at the top or the bottom  of its orbit.)
• If you have not done so, block the brain. (If your brain has been stored in the ultra cold freezer, allow it to warm up in the cryostat chamber for 15 or 20 minutes before attempting to block it.)
• Put a dab of embedding compound on the center of the groooved side of a specimen disk.
• Place the block (usually, posterior face down) on the embedding medium and place the specimen stage on a heat sink in the bottom of the cryostat chamber and allow the tissue to freeze to the specimen stage.  Embedding medium  will turn white.
• When the embedding medium has frozen, place the specimen disk  on the specimen head. 
• Set the object temperature on the control panel 1. Press the select menu key until OT flashes. The   Object Temperature  is usually kept at around -14° C. For fresh frozen tissue, the object temperature should be -10° to -14° C. For perfused tissue, set the object temperature a little colder (-14° to -18° C.)  Unlike its predecessor, the object temperature control is used to cool rather than warm the tissue.
• Two settings for sectioning thickness can be entered: one for trimming and one for collecting sections. The controls are located on control panel 2. To set the trimming thickness press the Trim/Sect button until the Trim light is lit.  The adjust the thickness with the + and - keys. The thickness indicator for trimming is located abve the Trim light on control panel 2. To set the Sectioning thickness, press th Trim/Sect button until the trim light is off. Adjust the tissue thickness using the + and - keys.  The  Sectioning Tissue thickness indicator is located on control panel 1.
• Turn on the light in the cryo-chamber.
• Using the Coarse feed controls,  begin advancing the tissue towards the knife. Arrows indicate direction of movement. Double arrow controls move the stage head more quickly than single arrow controls.
• When the tissue gets close to the knife, you may notice that you want to change the alignment of the tissue with the knife.  To do this, raise the lever located to the right of the specimen disk.  Now you can adjust the specimen orientation using the horizintal and vertical knobs located to the left and above the specimen disk. Reorient the tissue, then return the clamping lever to its original position.
• Begin taking sections using the hand wheel. Gently lower the glass anti-roll plate until its edge is laying along the knife edge. Sections should come off flattened between the anti-roll plate and the knife. Alignment of the anti-roll plate is critical to taking good sections. Take the time to optimize the alignment.
• There are a number of ways to collect the sections onto microscope slides. The easiest way is to carefully lower a warm microscope slide until it just touches the section and allow the section to melt onto the slide. If you use this method, it is wise to move the section to a part of the knife that is not cutting the tissue. This will prevent the build up of frost on the cutting edge. Tissue can also be picked up with cold forceps and placed on the slide. Perfused tissue can be collected with a brush, floated in distilled water or phosphate buffer and mounted on a slide using a brush. Perfused tissue must be mounted on gelatin coated slides !

Automatic Sectioning Mode

The Leica cryostat come equipped with a special feature... the automatic sectioning mode.l . Automatic Sectioning allows you to control the speed of the cutting stroke. This results in less distortion and more uniform sections. The controls for the automatic sectioning are found in the top portion of  of the control panel2.

• Turning handwheel clockwise, position the lower edge of tissue block about 2 mm above the knife edge. Press the button with the two arrows (above the V_MAX button) and hold until it blinks.
• Continue turning the handwheel clockwise until the upper edge of the tissue block is about 2 mm below the edge of the knife. Press and hold the button with the two arrows until it blinks. The cutting window is now set.
• Select cutting mode 1, 2, or 3 by pressing the appropriate button. Cutting mode 2 is for trimming tissue. When you press the foot peddle it will keep on sectioning until you press the foot peddle again. If a number has been entered into the preset counter on control panel 1, sectioning in mode 2 will stop when that number of sections have been taken. Sectioning mode 3 is for collecting sections. When you press the foot peddle a single section will be cut and the tissue stage will stop below the knife, out of the way to allow you to collect the section. In sectioning mode 1, the cutting will continue as long as the foot peddle is depressed.
• If you prefer, you may simultaneously press the "run enable" and  "run stop" keys on control panel 2 in lieu of using the foot pedal.
• Pressing the VMAX button will result in sectioning at full speed , ignoring the  cutting window.  Press VMAX again to resume with cutting window.
• The slide control on the right of the panel controls the speed of sectioning within the window.
• If you press hard on the foot peddle or press the red knob on the control panel, you will engage the emegency braking system. You must release the red knob (if depressed), press the "run enable" button, and reselect the sectioning mode (1,2,or 3) before you can continue sectioning.

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  Steve Milway
  Last modified: Wednesday, July 21, 2010 2:30 PM