The key to using para-phenylenediamine is pH - if the pH is below ~8.0,
you will see fading and background. We buffer our medium with
bicarbonate to keep the pH up.

The glycerol you use makes a difference, too. Some glycerols will
autofluoresce...I have *no* idea why, but we found a reliable source
(after testing glycerol from several vendors) but always check a new
bottle before using to make mounting media.

My own observation as far as Vectashield goes - it is fine if you
are looking at freshly prepared samples, but we start to see a lot of
media autofluorescence once the slides have been stored at -20C. Slides 
mounted with the para-phenylenediamine medium can be stored at -70C for
years and still be gorgeous on the 'scope. Prolong stores better than
Vectashield (in my experience) - but I'm convinced that it is
para-phenylenediamine-based, just from the way the dry ingredient looks 
and behaves.

My $0.02

Tamara Howard
CSHL

On Wed, 15 Dec 1999, Johan [iso-8859-1] Wassélius wrote:

> Dear David and fellow confocalists,
> 
> I have not used a wole lot of other mounting medias. What I have used a lot
> is Vectashield and another "home-brew" based on glycerin with
> 1,4-phenylenediamine (which is toxic). With the latter I see exactly what
> you mention i.e. a background caused by the antifading agent (especially if
> it takes a couple of days between the mounting and the examination of the
> slide). 
> 
> With Vectashield or PVA-DABCO I have subjectively not noticed any
> particular background introduced by the mounting medium, compared to simply
> mounting the slide in buffer and examine it right away. However, it does
> not mean that there is no background whatsoever. If your application is
> more sensitive then mine, you might find problems in situations where I am
> totally happy, I hope that is not the case here. 
> As for other mounting medias such as ProLong and others, I am sorry that I
> cannot be of much help for you. 
>