Re: [Histonet] Users of Prolong Gold Antifade from Invitrogen

From:"Gayle Callis"

We never let out sections dry out before applying Prolong Gold antifade 
mounting media.  Blot the excess liquid from around the section and apply 
the drop directly over the section.  If too much PBS is removed, we have 
problems with bubble formation. We are very generous with the drops, even as 
expensive as this media is.

We interpret removing excess liquid the same as blotting excess buffer from 
around a section during a staining protocol.

I suggest you contact their tech services (Molecular Probes site of 
Invtirogen) and ask them this same question.  They are prompt and very 

We have used Prolong Gold with DAPI on DSred label of fresh tissue (unfixed) 
frozen section, air dried for 30 minutes, then coverslipped.  We did examine 
the section the same day and had success but this is with DSred, very 
similar in structure to GFP and NOT a fluorophore e.g. Alexa dyes, FITC, 
TRITC etc. etc.   We had to do this since the DSred was compromised by loss 
for fluorescence due to either acetone or our favored acetone/alcohol 
fixation and autofluorescent issues with aldehyde fixatives.  The latter is 
a problem with GFP too.

Montana, USA has low humidity too, particularly in the winter, very dry.
Gayle M. Callis

----- Original Message ----- 
From: "Kevin Clayton" 
Sent: Wednesday, August 13, 2008 12:10 PM
Subject: [Histonet] Users of Prolong Gold Antifade from Invitrogen

> Hey all
> Anyone out there know how dry your samples (sections/cells) should be 
> before
> mounting using Prolong Gold? I have noticed (rarely) that sometimes all
> fluorescent signal - including DAPI - is much greater in some specimens 
> than
> others from the same slide and otherwise treated the exact same. The 
> product
> guidelines says to "remove any excess liquid from the specimen" but how 
> much
> excess is excess? I generally try to remove liquid drops and letting 
> visible
> liquid evaporate - without actually drying them - but a balance is 
> difficult
> to achieve and it is impossible to get all samples from an experiment to 
> the
> same level of "dryness". (And, man, do things dry quickly here in Spain
> compared to Ireland!) There's generally no problems with my samples but 
> the
> concern is that the fluorescent signal/background could be affected, which
> would be a big problem when comparing signal strength within experiments.
> If someone knows that would be great (and it would save me the obvious and
> fairly simple test I should really have done ages ago!).
> Thanks,
> Kevin
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