[3dem] Chemical fixation before plunge freezing or HPF

[Paul Verkade]

Hi Linda,

Indeed 4% PFA should work but we have also used  mixtures of 2%PFA with up to 0.5%GA. When working with pathogenic material we have chosen 2%GA to make sure.
GA should in general preserve ultrastructure better but for cryo-CLEM has a lot of autofluorescence.

The buffer and not leaving it in the fixative too long (i.e. freeze soon after fixation) are may be even more critical.

Best,
Paul

PS Hope Hugh is doing well
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On 18 Jan 2021, at 11:18, Linda Sandblad <linda.sandblad at umu.se<mailto: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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