The lab is equipped with a sliding microtome,
a vibratome,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.
Procedure
Place a sharp knife into the knife holder and secure in
place. Allow sufficient time for the knife to cool.
Using the horizontal motor drive on the control panel
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 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 Histo Prep ® on the center of the
groooved side of a specimen
stage.
Place the block (usually, posterior face down) on the Histo
Prep ® and place the specimen stage on a heat sink in the
bottom of the cryostat chamber and allow the Histo Prep (and brain, if
perfused) to freeze. Histo Prep will turn white.
Line up the red dots on the specimen stage with the red
dots on the tissue
clamping device. Press the lever above the stage and turn the
stage right or left until a red mark on the stage is located between
the green marks on the tissue clamping device. Release the lever.
Set the object temperature on the control panel.
The object temperature is always warmer than the box temperature which
is usually kept at around -24° 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.)
Using the dial located above and to the right of the
specimen stage, select the section thickness (0 to 60µm).
Turn on the light in the cryo-chamber.
Using the horizontal motor drive begin advancing the tissue
towards the knife. When the tissue gets close to the knife, the light
will cast a shadow of the knife edge across the face of the tissue
block. By moving the tissue up and down with the hand wheel and
watching the shadow, you will be able to tell how well the tissue is
aligned with the edge of the knife. If the tissue is not well aligned,
release the clamping lever on the tissue clamping device by moving
it from vertical to horizontal. Reorient the tissue, then return the
clamping lever to the vertical 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. Like all special features, this
one is prone to all manner of technical problems and often doesn't
work. When it is working it 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 box of the control
panel.
Turn on the green button located under the red knob.
Turning handwheel clockwise position the lower edge of
tissue block about 2 mm above the knife edge. Press the button with the
down arrow to the right of the green 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 up arrow to the
right of the VMAX button 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. 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.
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 green button, and
reselect the sectioning mode (1,2,or 3) before you can continue
sectioning.
No, I don't know why a feature that is so rarely
operational has two emergency "brakes" ... unless
they are "breaks"...that would explain a lot.Steve
Milway
Last modified: Friday, February 21,
2003 3:17 PM