Hi,
I have some ctyospins of cells exposed to fluorescent nanoparticles, I'd also like to stain the slides with DAPI to prepare for confocal fluorescent microscopy. Is there an advantage to staining with a DAPI solution, then mounting with a non-quneching medium vs using a mounting medium with DAPI in it already?
How well do the mounting media containing DAPI work when checking for fluorescence? Is there a huge difference between using a hardening vs non-hardening medium?
Hi Ariel, most mounting media is a glycerol solution with trace antioxidants to prevent air oxidation of fluorophores. That's why they are so cheap.
A lot of companies sell mounting media with DAPI because DAPI/DNA fluorescence is almost impossible to bleach in either regular or confocal fluorescent microscopy.
Don't bother with hardening/non-hardening stuff. If your cells are fixed then glycerol containing mounting media is fine.
Hi Steingrimur,
Thanks for your response!
As far as I can tell, the advantage to using a mounting medium without DAPI in it is that then it's easier to tailor the concentration of the DAPI stain.
This was the mounting medium I was looking into, containing 1.5 ug/mL DAPI:
http://vectorlabs.com/vectashield-mounting-medium-with-dapi.html
My cytospins were also fixed in methanol. I've read from a product data sheet from the company above that for cells fixed in methanol non-hardening mountant was preferred because the DAPI would diffuse out of the nuclei over time.
Hi Ariel, I can guarantee that once DAPI stains DNA, it will be there forever.
My concerns would be about the methanol treatment. Methanol dehydrates the cells and solubilizes the lipids but it is not a true fixative. Treat adherent cells with methanol and they stay there dry- no problem. Add aqueous solutions to them and you see cell structures melt away before your eyes.
Fixatives, like formaldehyde and Glutaraldehyde, crosslink all the amine containing molecules together so that cell structures are fixed. Your cytospin methanol solution must have some formaldehyde in them.
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