Immunofluorescence photobleaching, is mounting medium to blame? - (Jul/10/2013 )

We've recently been having some problems with bleaching of our samples when viewed at confocal, it has started to happen in more than one channel at once so I'm thinking that it's not a problem with a secondary (unless they've all gone off at once!). I was wondering if the mounting medium might be to blame, I've used vectashield for years and never had problems with it but the bottle I'm using has been open for a while and is past its use by date - has anyone ever known mounting medium to "go off"?

The samples I'm talking about are PFA fixed frozen rat spinal cord and unfixed frozen DRG either double or triple labelled using Alex Fluor secondaries.

Any other suggestions would be welcome too, thanks

Don't know about the vectashield, but commonly photobleaching on the confocal is due to having the lasers at too high a power. In my experience (admittedly only on ICC cells) there is a bit of a trade off between using a high gain and laser power. I prefer to have the gain set about 20-30% with medium to low power on the lasers.

Interesting you should say that because I had wondered if it was something to do with the microscope - especially since it's been out of action since I returned from a break. I shall investigate further, thanks!