I was cryo-sectioning fixed-frozen and fresh-frozen mouse brains, and found a lot of wrinkles and folding in the fixed-frozen sections, while the fresh-frozen sections were pretty smooth. Below are my sectioning conditions and observations. I would appreciate any advice on improving the fixed-frozen sections.
Tissues: fixed-frozen mouse brains (OCT embedded), fresh-frozen mouse brains (OCT embedded).
Tissue temperature: at -80 deg for at least 48hrs, equilibrated to -18 deg for 1hr before sectioning.
Thickness: 14um
Slide: Superfrost plus
Cryostat chamber and head temperatures: -18 deg, -21 deg respectively.
Observations: When the fresh-frozen sections came out of the block, they rolled up smoothly, “jumped” to the slide immediately, and unrolled themselves smoothly. But the fixed-frozen sections looked more “limp” – they were “wavy”, could not roll up, and when I put a slide over them, they could not extend smoothly thereby creating wrinkles, folding and even bubbles. I have attached an image showing my fresh-frozen sections at the top, and fixed-frozen at the bottom.
It looked as if the fixed-frozen block was “warmer” than the fresh-frozen block, but in fact they had been equilibrated to -18 deg for the same amount of time (~1hr). And I believe it was also not a blade artifact, because I alternated between cutting fixed-frozen and fresh-frozen sections several times using the same blade. The fresh-frozen sections looked fine each time, but the fixed-frozen sections were always problematic. I also believe there was no temperature difference between the slides used for fixed- or fresh-frozen sections (both were stored at room temperature).
My protocol for preparing the fixed-frozen tissues is listed below:
1. Transcardial perfusion of PBS (10ml) and 4% PFA (10ml). 2. Fixation with 4% PFA at 4°C overnight (~18hrs). 3. Wash with PBS (20min x 3). 4. Dehydration with 15% sucrose (in 1X PBS) at 4°C until tissue sinks (~12hrs). 5. Dehydration with 30% sucrose (in 1X PBS) at 4°C until tissue sinks (~24hrs). 6. Embedding in OCT with dry ice and 100% EtOH until OCT solidifies. 7. Stored at -80°C.
What do you think I could do to improve the fixed-frozen sections? Your input would be much appreciated.
Thanks for your response, Matthew Ibbs .
So for 1., my fresh-frozen tissues are snap-frozen in liquid nitrogen, and then embedded in OCT using the same procedure as for the fixed-frozen tissues (on dry ice + EtOH).
2. The ethanol is mixed with dry ice to create a cooling bath. I didn't add ethanol to the tissue or to the OCT directly.
3. This is a good point. I agree with you that the temperature during fixation could make a difference, and it would be worthwhile to test difference fixation schemes for our particular tissue type.
Your further comments are more than welcome.
Just sharing what worked for me for any future seekers! To avoid folding/wrinkling/bubbles in cryosections- What I do is keep my (newly purchased fisher) super frost plus slides inside the cryostat (-18C gives smoother sections) and let them cool while I section the brain (I do 10um thick sections). Once the section is cut with the anti-roll plate over it, I flip the section, flatten it then scoop onto my cold slide. The section does not attach since both are cold and gives this advantage of having more control on the attachment. Then I use my brush to flatten the section perfectly on the slide and use my warm finger under the glass slide to evenly melt the OCT onto the slide. I dry using a fan for ~1hr and then keep at -20C before putting at -80C. I have been getting perfect adhesion and even mounting!
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