I have some PFA-perfused brain tissue. I post-fixed with 4% PFA and put into 30% sucrose and snap-freeze for cryostat. Now I want to use vibratome, so I slowly thaw them and put into PBS for vibratome. I am using 0.5mm/s for speed and 1.35mm for amplitude (50um thickness), but the tissue tends to roll up or tear when cutting. How should I adjust speed or amplitude?
Jiabin Tang
Hi there! I'm Abby, Chief Scientific Officer at Precisionary Instruments, where we've been crafting Compresstome vibratomes and microtomes for nearly two decades. With a wealth of experience in tissue sectioning, I'm here to help!
When you use a vibratome to section PFA-fixed tissue, you don't need to soak it beforehand in 30% sucrose. The sucrose soak is primarily intended to protect your tissue for freezing temperatures on the cryostat.
To prevent rolling or tearing of your sections, consider decreasing the speed. Additionally, for fixed tissue, it's worth noting that higher oscillation or frequency of cutting may not necessarily be advantageous. In some cases, decreasing the oscillation can yield better results.
One key trick for obtaining perfect fixed 50µm thick sections is to embed your tissue sample in agarose. Agarose embedding stabilizes your tissue sample during the cutting process and prevents it from rolling. You can see how this works with our patented Compresstome vibratome by following this link: https://precisionary.com/tissue-sectioning-help/how-is-compresstome-different/
Best regards,
Abby Chu, MD-PhD
CSO
Precisionary Instruments
Firstly, full disclosure - I make vibratomes!
Assuming you are following a standard protocol for thawing, then 50µm sections from fixed tissue should be easily achievable with a reasonable vibratome. Agarose embedding can help with curling prevention, but should not be necessary.
I would certainly reduce the speed as that is quite high, and reducing amplitude should help with delicate samples. What blade frequency are you using?
However, I would also look at the blade you are using. Don't let anyone convince you that a standard razor blade is as good as a purpose made stainless steel blade or ceramic blade. Razor blades are relatively blunt, designed to cut hair and not skin, and introduce compression (I am not saying this for any commercial reason, it is fact!).
If you have the opportunity to use fixed tissue that won't have had the cryostat treatment, you could ascertain whether the freezing and thawing regime is the problem.
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