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Roberto,
Vectashield (hard or aqueous) dramatically quenches/diminishes fluorescence
from Alexa-647 (>10-fold quenching from my own experience). A similar
problem frustrated me, and it amazes me that there is not mention of this
limitation on the Vectashield insert. I have since switched to the Life
Tech/Invitrogen Prolong (hard) or Slowfade (aqueous) mountants, which do
not appear to affect far-red fluorescence from Alexa647.
Josh Meisner
Department of Biomedical Engineering
University of Virginia
On Mon, Jul 16, 2012 at 10:34 AM, Roberto Fiorelli <
[hidden email]>wrote:
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>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> Hi all,
>
> I used to inject EdU to detect proliferating cells of the neurogenic
> regions
> of the brain. The nuclear incorporation is then detected with the
> Invitrogen
> kit Click-it, where a catalytic reaction links the alexafluor-conjugated
> azide to the EdU. The results are excellent if I use Alexa-555 azide, with
> a
> bright stable staining (up to 6 months after), with the sections mounted in
> Vectashield hardset. Instead, when Alexa-647 is used, I experienced huge
> problems with the signal fading as short as after 2 days from mounting. I
> actually suspect this is due to leaking rather than bleaching. Obviously,
> right before mounting the staining looks good and specific. But after only
> two days, it's completely gone.
>
> Has anybody experienced the same ind of problem?
>
> Roberto
>
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