Kubke Lab:Protocols/Cryomicrotomy

 WORK IN PROGRESS --- EXPECT FREQUENT CHANGES

Cryostat sectioning

Equipment:

The cryostat available through the Histology Facility at the University of Auckland is a Leica – CM3050S. The razor blades used to cut were Leica – Low profile blades.

• Hold the key button to turn on

The cryostat is always turned ‘on’ but holding this button will allow the interface to be displayed and reached

• Unlock the hand-wheel to use by moving the locking pin on the hand-wheel towards the cryostat

Until this is done you will not be able to change the settings on the cryostat

• Blades should be kept in the freezer compartment so that they are at the same temperature as the block and the chamber
• Ensure the blade is sharp and lacking in nicks. Nicks will cause crinkling in confined areas in the sections and will reflect the location of the nicks
• Align the blade to the platform at 5 degrees
• Ensure the TRIM light is OFF
• All cryostat accessories including the metallic forceps, paint brush (best with stiff bristles) should be kept in the freezer compartment
• Wear safety goggles, a lab coat and gloves

Making a mould:

1. Place a small amount of OCT (Optimum Cutting Temperature by TissueTek MSDS) on the bottom of the plastic mould and for heavier tissues allow the OCT to freeze slightly before adding the tissue
2. Lay the tissue flat on your semi-frozen OCT surface and then completely cover the specimen in OCT
3. Place the mould into the cryostat chamber to freeze.

OCT is ‘hard’ when it has turned its maximal white colour

Mounting onto the metallic chuck:

1. Place a small amount of OCT on the chuck such that the grooves of the chuck are covered but not so much that the tissue mould is protruding too far from the chuck; this will increase the chance of it breaking during trimming
2. While the OCT is freezing hold the tissue mould onto the chuck so that it becomes frozen within the chuck’s OCT

• Attempt to make the OCT structure one continuous piece to help prevent the chance of a fracture occurring within the OCT
• Ensure that there is sufficient OCT supporting the base of the tissue mould and sufficient on the 'top' of the mould (the end that is reached last by the blade) since this makes picking up the sections easier.

Cutting:

• Make sure that the front end of the tissue mould is 'behind' the edge of the blade before lowering to cut. Advance slowly until the first sections can be cut.
• Slowly advance the specimen, until you start getting sections. Continue until a nice cutting surface has been formed.
• Trim the face of the block to a nice shape
• Speed of cutting may need to vary depending on tissue type, temperature, block size, etc.
• After lifting each section, clean the blade with the brush.
• Try to keep the chamber's door closed as much as possible.

Lifting sections onto the glass slides:

• When mounting the slides attempt to have the section ‘jump’ onto the slide as opposed to a ‘roll’, since rolling the specimen onto the slides may cause damage to the histology
• Each section should be picked up individually.

Using the microscope:

You should check the integrity of your sections under a microscope regularly during your sectioning. This may indicate potential problems and may give clues as to how to eradicate the problems and what factors to adjust in your setup.

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