[Histonet] Improving wrinkles and bubbles in fixed-frozen mouse brain cryo-sections?

[Anna Xiao Luo]

Dear List,



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.

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 as follows:

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.



Thank you,

Anna Luo

PhD Candidate

Department of Neuroscience

Johns Hopkins Medical Institutions