Mounting Media or Anti-fade Media
Mounting media or Anti-fading media plays three important roles in microscopy:
1) Refractive index matching to reduce optical artifacts
2) Anti-fade to prevent photo bleaching of fluorophores
3) Preserve structure of cell.
- Polymerizing reagents do not preserve the cell structure well, i.e. the cells will flatten when the media sets.
- I do not recommend mounting media with embedded stains, e.g. such as dapi.
- Many advanced imaging labs make their own mounting media or anti-fade media for specialized imaging such as super resolution. Check literature and online forums.
There are several types of mounting Media or anti-fade reagents on the market.
Dako Fluorescent Mounting Media
- Polymerizing, good for routine wide field imaging or slide scanning, $
- Many levels of quality, for high quality confocal imaging, $$ – $$$
- WARNING, can quench certain fluorophores, read technical notes carefully!
- Vector Laboratories
Most objectives are designed to image through glass that is 0.17 mm thick, as such be sure to buy good quality number 1.5 thickness cover glasses. This is especially important for confocal and TIRF imaging.
LCI – Chamilide Chambers
Glass Bottom Dishes
- Can be costly over time as they are not reusable, $$
- Preferred if you study requires sterile conditions, if you are using reagents that are hard to clean between samples, or if you are working with biohazard and you need a sealed container
- Be sure that glass is high quality number 1.5 thickness
IBIDI – Imaging Chambers
Media for Live Cell Imaging
- Should NOT contain phenol red or serum
- pH Buffering
- HEPES buffer – does not require CO2 but can lead to production of reactive oxygen species
- Bicarbonate buffer – requires CO2, more gentle on cells