Hi all, I have to postpone some stainings and imaging due to technical difficulties, but the cells are already in culture. They are hiPSC derived cortical neurons in 96well plates and the staining will be Bodipy.
- how long can I keep them in the fridge after PFA fixation?
- is it better to stain first and keep them that way or stain freshly? The structure I'd like to stain is lipid droplets, so I'm leaning towards the first possibility.
Thanks for all comments
PFA doesn't fix lipids, so you'll probably see a gradual drop in the lipid content (with our without dye). In terms of overall structural preservation (TEM level) you can keep cells/tissues in PFA in the fridge indefinitely in a tightly sealed tube, top off with fresh PFA if needed so they don't dry out.
Thanks! That already helps. Have you fixed lipids in cell culture before and if so, what did you use? Thx
Lipids are pretty much impossible to fix. In electron microscopy you fix and stain lipids with osmium tetroxide (very toxic and dangerous stuff) but the majority of lipids are lost anyway. Your best bet with fluorescence is to image live, or use advanced cryopreparation methods (probably overkill). Think of it as imaging soap bubbles, best to be quick! If you fix with aldehydes you can only hope that by fixing the proteins you also stabilize certain lipid structures too, but the damage happens within minutes. Just keep that in mind, do a live control experiment and don't overinterpret your data.
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