[3dem] Chemical fixation before plunge freezing or HPF

[Amar Parvate]

Hi Linda
I have used 1% GA for fixing  BSL3 viruses for cryo-EM. Resultant single
particle resolutions ranged between 7-9 angstrom. A have also used 2% GA
while fixing BSL3 virus infected cells for HPF followed by thin section
TEM. If you are fixing mammalian cells intended for HPF, i would very
strongly recommend not centrifuging them for >500g at any time during
fixation or further washes, even ~200g if you can go that low. GA helps
preserve ultrastructure for sure but I would recommend leaving your sample
in the fixative only for the minimum essential amount of time. There are
several reports out there with time recommendations.

If you need further details of the protocols here are links -
1) https://urldefense.com/v3/__https://www.frontiersin.org/articles/10.3389/fcimb.2020.580339/full__;!!Mih3wA!TMDW6o_cpZIwgUlq8Ekweus0ftgX0bj6FrhPvLkrP1M30CrZe6d64_bZErBdSi4cVA$  - *for
HPF*
2) https://urldefense.com/v3/__https://www.sciencedirect.com/science/article/abs/pii/S0166093419304173__;!!Mih3wA!TMDW6o_cpZIwgUlq8Ekweus0ftgX0bj6FrhPvLkrP1M30CrZe6d64_bZErBQcnm7Kw$ 
- *for inactivation of viruses intended for cryo-EM/cryo-ET*

P.S. - I have never used PFA and I have never done CLEM so I dont know how
that would affect either fixation or fluorescence experiments.

Good luck!
Amar


On Mon, Jan 18, 2021 at 3:19 AM Linda Sandblad <linda.sandblad at umu.se>
wrote:

> Dear best colleagues!
>
> What chemical fixation protocols works for your projects before plunge
> freezing (or HPF) or what typs of chemical fixation was not good (destroyed
> more than it helped) prior to cryo-EM sample preparation?
>
> Im interested in your experiences, also happy if you have adressed this
> question in a article and would like to share the link with me.
>
> For cryo-EM we prefer to work as close to native as possible, avoiding
> chemical fixations, but sometimes a fixation is needed to test or move on
> with tricky projects. For examples, when bacteria cultures are tricky and
> samples need to be stored, or when bio safety restricts work in the EM labs
> (lab specific and different).
>
> Best regards, Linda
>
> Linda Sandblad, researcher and facility director
> Umeå Core Facility for Electron Microscopy, SciLifeLab Cryo-EM, NMI
> Department of Chemistry, Umeå University, Sweden
> Tel: +46 709324936
>
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>


-- 
Amar Parvate
Saphire Lab
LJI
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