Cryostat Article #6: Troubleshooting Cryostat Tissue Sectioning Problems

Cryostat Article #6: Troubleshooting Cryostat Tissue Sectioning Problems

Anyone working with a cryostat to make thin frozen tissue sections will have encountered some sort of obstacle. Issues like tissue cracking, freeze artifacts, and curled tissue slices are commonly experienced but can be easily troubleshooted. In this week’s article, we would like to help focus on solving common problems associated with cryostat-produced tissue sections for research and clinical studies.

Common cryostat problems (& how to fix them!):

  • Tissue folding or curling. This is one of the most common problems! Basically, frozen tissue slices curl as it is cut. To battle this problem, use a fine tissue paintbrush to gently flatten the curled tissue. Make sure that your cutting blade is sharp, and it may help to change out your cutting blade. You can also use an anti-roll plate, which is a glass slide that fits right over the cutting stage, and allows the cut tissue to stay flattened as it is cut.
Figure 1. Frozen tissue sectioning may result in unwanted curled tissue sections (A). This can be remedied by using a fine tissue brush (B) to slowly maneuver the tissue as it is being sliced (C). An anti-roll plate is also recommended to prevent tissue curling (D).
  • Tissue cracking. Cracked tissue is usually from over-freezing the embedded tissue sample. For cryostats that have two compressors, both the freeze chamber and the specimen holder temperatures can be controlled. How to solve this problem? Check your cryostat temperature and make sure it’s not too cold. Adjust the temperature by increasing it 5-degree until you no longer get tissue cracks in your sections.
  • Tissue smudges or streaks. If you see line streaks vertically across your tissue slice, check your cutting blade carefully. Sometimes frozen debris or OCT is stuck on the blade and can cause streaks. Clean the cutting blade, or move the blade to a new section for cutting. Sometimes you may need to replace the blade.
Figure 2. Frozen tissue cut on the cryostat may have unwanted streaks and cracks (left), freeze artifacts (middle), or smudges and chopped sections (C).
  • Tissue sticks to the brush or rips. This usually happens because the user is pulling the frozen tissue slice. When you use a fine tissue brush, make sure to roll the brush instead of tugging. In addition, make sure you are using a tissue brush with fine bristles, and that the tissue slice is not getting caught between the bristles.
  • Tissue is not sticking to the glass slide. When you are ready to mount your frozen tissue section, you can take a glass slide and touch the surface to the tissue. The tissue will usually adhere to the glass slide and the OCT will melt. If you are not getting this result, it could be that the temperature of the glass slide is too low, or the glass slides have been kept inside the freeze chamber. The temperature difference is what allows the tissue to stick to the slide when you try to mount it on glass.
  • Chuck (specimen holder) is stuck. Oh no! If the chuck you are using gets stuck on the specimen holder clamp, use 70% ethanol to clean and unfreeze it. The stuck chuck is usually caused by small amounts of water being trapped inside the specimen holder clamp. You can also run a defrost cycle to unfreeze the chuck.

Precisionary Instruments is here to help you with cryostat sectioning! Always remember to use the safety lock on the hand wheel when you are adjusting anything inside the freeze chamber. Contact us with any questions, and explore our brand new Precisionary Cryostat CF-6100!

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