Cryostat Article #5: Cryostat Frozen Section Technique

Cryostat Article #5: Cryostat Frozen Section Technique

How do you make the “perfect tissue slice” on a cryostat? Although we can’t be there with you in person, we hope to offer some detailed and helpful tips for how to obtain superior sections with the Precisionary Cryostat CF-6100. This week, we would like to describe step-by-step instructions for how to prepare frozen sections. Then we will discuss tips & tricks for fixing common problems encountered in cryostat use.

Preparing Tissue for the Cryostat: Tissue Freezing Methods

The tissue you want to slice should be frozen or fixed as soon as possible after dissection. This is to help avoid tissue drying artifact and autolysis (destruction of tissues or cells by enzymes). Why do we want to snap freeze tissue or fix tissue and then cryoprotect it before freezing? It’s because slow freezing can cause distortion of tissue due to ice crystal formation. This causes a “Swiss cheese” effect (Figure 1) and introduces freeze artifacts into tissue.

Figure 1. Freeze artifacts in frozen sections of mouse spleen with H&E to review morphology. Left image is a normal tissue slice that was correctly and rapidly frozen. Right image shows significant freeze artifacts, and results cannot be used.

There are two commonly used methods of tissue freezing:

  1. Fresh tissue freezing: Tissue is embedded in OCT media and flash frozen fresh.
  2. 4% PFA fixed, then cryoprotected: Tissue is fixed first, then soaked overnight in 30% sucrose, then embedded and frozen.

Making Tissue Sections on the Cryostat

The optimal temperature for cryostat sectioning depends on the type of tissue and on whether the tissue samples have been freshly frozen or pre-fixed and then cryoprotected.

Tips & Tricks

  • Tissue brushes: The purpose of the brush is to grab and maneuver the tissue section across the stage. This helps give you additional fine control of sections being sliced (Figure 2).
  • Anti-roll plate: This apparatus is used to prevent frozen sections from curling after being cut. The anti-roll plate is aligned parallel to the knife edge, but a little above it. You can adjust the anti-roll plate’s height and angle, so that a slice being cut can easily be guided onto the cutting stage and below the anti-roll plate (Figure 2).
  • Mounting tissue sections: Once frozen sections are cut, they can be either directly mounted onto a glass slide (the tissue and OCT will melt onto the glass and adhere directly). Or, you can use a tissue brush to pick up the tissue slice and store it in buffer as free floating sections to be mounted onto glass slides later (Figure 3).
Figure 2. Left image shows how a fine tissue brush is used to grab a tissue section across the stage. Right image shows an anti-roll plate helping keep a newly cut frozen section flat.
Figure 3. Left illustration shows how cut frozen tissue using an anti-roll plate can be directly mounted onto a glass slide. Right image shows a glass slide touching a tissue slice, causing it to adhere to the glass.

As we continue with our new Precisionary Cryostat CF-6100 launch, we will keep on posting helpful articles!

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