DAPI weirdness with hardset Vectasheild

[simon walker (BI)-2]

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Has anyone else observed weird behaviour of DAPI when used in conjunction
with hard-setting Vectasheild?  When exposed to wide-band UV excitation we
have seen the DAPI excitation characteristics change such that the DAPI
becomes excitable with 488 nm light.  And today someone showed me a slide
where (again using wide-band UV excitation) the DAPI signal initially looked a
pinky/red colour, but after a few seconds of exposure developed the usual
blue fluorescence.  We don't seem to have these problems with the liquid
mount Vectasheild.  Any suggestions as to what might be going on??
Thanks,
Simon

[Neil Kad]

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Simon,

Can you check the spectral properties using a spectrometer under the different conditions?

Cheers

Neil

> Date: Fri, 7 Oct 2011 05:51:54 -0500
> From: [hidden email]
> Subject: DAPI weirdness with hardset Vectasheild
> To: [hidden email]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Has anyone else observed weird behaviour of DAPI when used in conjunction
> with hard-setting Vectasheild?  When exposed to wide-band UV excitation we
> have seen the DAPI excitation characteristics change such that the DAPI
> becomes excitable with 488 nm light.  And today someone showed me a slide
> where (again using wide-band UV excitation) the DAPI signal initially looked a
> pinky/red colour, but after a few seconds of exposure developed the usual
> blue fluorescence.  We don't seem to have these problems with the liquid
> mount Vectasheild.  Any suggestions as to what might be going on??
> Thanks,
> Simon

     

[Kelly Lundsten-2]

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The red shift (emission in the green) is due to over labeling with the DAPI.  When you illuminate, thus photobleaching some of the individual DAPI molecules, the proximity effect is relieved and it emits blue again.  So, question, is the DAPI contained in the mounting medium?  If not, halve the concentration of DAPI (keeping incubation time consistent) and you won't force the individual molecules to be so close together.  I normally choose not to mount with DAPI in the mounting media, due to this abberration and often seeing collisional quenching of the DAPI at the center of the nucleus overtime in addition to the red shift.  Both conditions due to over labeling, both relieved with photobleaching.

Kelly Lundsten
BioLegend
Business Segment Mgr, Adv. Cytometry
773-633-4774

On Oct 7, 2011, at 3:32 PM, "Neil Kad" <[hidden email]> wrote:

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> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
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>
> Simon,
>
> Can you check the spectral properties using a spectrometer under the different conditions?
>
> Cheers
>
> Neil
>
>> Date: Fri, 7 Oct 2011 05:51:54 -0500
>> From: [hidden email]
>> Subject: DAPI weirdness with hardset Vectasheild
>> To: [hidden email]
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Has anyone else observed weird behaviour of DAPI when used in conjunction
>> with hard-setting Vectasheild?  When exposed to wide-band UV excitation we
>> have seen the DAPI excitation characteristics change such that the DAPI
>> becomes excitable with 488 nm light.  And today someone showed me a slide
>> where (again using wide-band UV excitation) the DAPI signal initially looked a
>> pinky/red colour, but after a few seconds of exposure developed the usual
>> blue fluorescence.  We don't seem to have these problems with the liquid
>> mount Vectasheild.  Any suggestions as to what might be going on??
>> Thanks,
>> Simon
>                        

[George McNamara]

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Yes, I have seen DAPI, as well as Hoechst (33342, 44258) photoconvert to
green. This has been previously discussed on the listserv.

To avoid this, try using a much lower concentration, incubate the
counterstain, then wash, then apply mounting medium. Avoid using old
mounting medium. Change to Prolong Gold (without DAPI).



On 10/7/2011 6:51 AM, Simon Walker wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Has anyone else observed weird behaviour of DAPI when used in conjunction
> with hard-setting Vectasheild?  When exposed to wide-band UV excitation we
> have seen the DAPI excitation characteristics change such that the DAPI
> becomes excitable with 488 nm light.  And today someone showed me a slide
> where (again using wide-band UV excitation) the DAPI signal initially looked a
> pinky/red colour, but after a few seconds of exposure developed the usual
> blue fluorescence.  We don't seem to have these problems with the liquid
> mount Vectasheild.  Any suggestions as to what might be going on??
> Thanks,
> Simon
>
>    

--


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami