Hi, I am taking over a project that has many interesting microscopy samples stored in PBS + sodium azide for various number of months or years. I want to know how old can immunofluorescence samples be without mounting media, and still get reliable image data?
The samples are from adherent cultured mammalian cells fixed with formaldehyde and permeabilized with triton. They are labeled by indirect immunofluorescence; dyes like phalloidin, Hoechst, and a fixable mitotracker; and EdU labeled by click reaction - I would imagine some of these will be more stable than others without a viscous mounting medium. The fluorescent labels include rhodamine, Alexa Fluor dyes, mitotracker deep red, and others - I would imagine some of these would lose their fluorescence without free radical quenchers and other additives of mounting medium. I could consider changing out the PBS in there currently for mounting medium and then imaging, since that would help reduce photobleaching during the imaging process, but won't fix the damage done during the storage time - do you think this would be worth it?
Thanks!
Hi Beverley Marie Rabbitts . The fluorophores are probably still OK if the samples were stored at 4oC and protected from light. I would be more worried about the integrity of the samples themselves.
Phalloidin staining should be a good indicator. It would be good if the staining still shows sharp actin bands.
Do you have images of the same samples taken earlier for comparison?
Hello Beverley Marie Rabbitts
The samples are stable and efficient enough to result correct data when you keep in PBS+NaN3 medium for months (4°C). But you should be very careful while handling NaN3 mixture, due to its death causing toxicity.
I hope this would be helpful.
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