Dear Nick Brose,

I recommend not to opt for fixed cells if you want to image lysosomes. In most cases, lysosomal localization is driven by pH. As PFA fixation impacts the pH, you will end up having non-specific interactions. Hence, always use live cells for lysosomal imaging. Nevertheless, the viability is out of the question here as cells will be dead upon fixation and you can use them for years depending on how efficiently they are fixed and stored thereafter.

Thanks

Dear Nick Brose

These articles might be useful, have a look:

1. https://www.frontiersin.org/articles/10.3389/fchem.2019.00588/full

2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2669416/

3. https://www.sciencedirect.com/science/article/pii/S2666166720301192

Kind Regards

Qamar Ul Islam

Hi Nick Brose

If you are interested in lysosomal distribution and localization, antibody staining (for example Lamp1is a good marker on lysosomal surface) is perfectly compatible with PFA fixation. You can fix your cells, perform an immunofluorescence for Lamp1 and store your preparation at 4ºC for as long as you want. As Tanoy Dutta was saying, cells are not alive so viability is not an issue. But in case you need to study lysosomal pH, you will need your cells alive indeed, so you won't have to fix them with PFA. There are available probes pH sensitive such as LysoSensor.

Hope this is useful!!