Protocol

Mounting media is cheap and easy to make yourself, and gives better results than many of the expensive commercial options out there. You only need three things from a fluorescence mounting media…it should buffer your sample and fluorophores, prevent photobleaching, and have a high refractive index.  That’s it!

Here’s a commonly used recipe we like:

  • 20mM (final concentration) Tris, pH 8.0. Many fluorophores are brighter at higher pH.
  • 0.5% N-propyl gallate. This stuff helps prevent photobleaching.
  • 50-90% Glycerol. Glycerol raises the refractive index of the mounting media, so you’ll get brighter and higher resolution images. The higher the glycerol concentration, the better the fluorescence image but the worse the DIC (Nomarski) image. If you are doing fluorescence only, use 90% glycerol (yes, it’s a pain to pipette – we promise it’s worth it!). If you need to take a DIC image as well, use 50% glycerol so you don’t lose the DIC contrast.
  • You will probably need to warm the solution to 37C and vortex to get it to go into solution.
  • Use ONLY about 6-8ul of mounting media per 18mm coverslip. The solution should slowly spread to the edges after you place the coverslip onto the media. If you add too much it will leak out the sides and prevent the nail polish from sealing the coverslip to the slide. If some solution does leak out the slides, carefully wick it away with a kimwipe or Whatman paper. Unlike some commercially available mounting medias, this media will not harden over time, so good sealing with nail polish is needed to preventing the slides from drying out.
  • Seal coverslip onto slide with nail polish
  • Store mounting media at 4C

Commercial Options

If you want to buy a pre-made mounting media, we’ve gotten good results with…

Use other commercial options at your own risk – there are some pretty bad ones on the market. Bad mounting medias scatter light, making your signal weaker and your background brighter. Tragic!

DO NOT use any mounting media with DAPI in it.  *gets up on soap box*. DAPI is a fluorophore with a broad excitation and emission spectra.  If it’s in your mounting media, you are adding background fluorescence to your sample!  Sure, it’s brighter when it’s bound to DNA. But it IS fluorescent when it’s not bound to DNA…floating around in your mounting media, making your background brighter and reducing your image contrast :(. We hate mounting media with DAPI in it. Really, really hate it. If you want to stain your cells with DAPI, by all means do!  Buy DAPI, dilute it as instructed, dip your sample into it, and rinse away the excess. Then mount in a nice, dark, fluorophore-free mounting media. Then tell your friends to do the same. (PS, the same goes for mounting medias with propidium iodide, or any other fluorophore).