Community collective: imaging the secretory pathway

Advances in labelling methods and imaging technology in the last 10 years have allowed us to fully appreciate the dynamic nature of the secretory pathway, especially the extensive remodelling of the endoplasmic reticulum (ER). However, preserving the ultrastructure of these dynamic structures requires specialised fixation methods, and in a recent tweet Guillaume Jacquemet asked for advice on preserving the morphology of ER and Golgi.

The top suggestion, from @raj_mito, @aaandmoore and @DamstraHugo, was to add a little glutaraldehyde. Interestingly, the use of glutaraldehyde to quickly fix ER was inspired by a paper published in 1984 by Mark Terasaki (thanks for the tip @aaandmoore!) Glutaraldehyde is often used in electron microscopy because it preserves the ultrastructure, but the crosslinking can affect epitope availability, meaning that your choice of fixation protocol will depend on the labelling method. This is true when looking at different structures in the cell, and you can read more in our primer from Heather Brown-Harding.

Andy Moore shared a movie showing the ER tubules freezing upon addition of glutaraldehyde.

Christoph Spahn @miCHRIScopy recommended using the protocol from Eric Betzig.

And how can you tell if the fixation is suboptimal? Look out for pearling or vesiculation of the ER. We reached out to Andy to find out the common causes of vesiculation and in his hands the top culprits were shear force from adding fixative with too much zeal (NB Andy recommends adding fixative to the media already covering cells rather than aspirating the media and adding fixative), and using a fixative in a solution is not osmotically balanced.

Update:

Stephen Royle shared his lab’s glutaraldhyde-free method of fixing the ER over on Mastodon.
“Our experience is also that ER fixation can be tricky. One glutaraldehyde-free method that we have used is fixing for 2 h in 2% paraformaldehyde in 87.5 mM lysine, 87.5 mM sodium phosphate, pH 7.4, and 10 mM sodium periodate.”

If you have requested (or seen) any community advice that you would like to have a permanent home on FocalPlane, contact us at focalplane@biologists.com or on our social media pages. And if you are not on social media but would like to solicit community advice, then drop us and email and we’ll ask on your behalf.

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