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Hi Dale,
You might find that paper from Melike interesting:
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0101772

They use sodium borohydride (NaBH4) to quench fluorescence before
restaining with the same dye (AF647) but against different cellular
structures.
I hope this helps.

Romain

-- 
Dr. Romain Laine, PhD in Biophotonics
Laser Analytics Group
Department of Chemical Engineering and Biotechnology
University of Cambridge
New Museums Site
Pembroke Street, Cambridge, CB2 3RA, UK
T: (+44)1223330133




On Thu, Feb 16, 2017 at 5:54 PM, Kelly Lundsten <[log in to unmask]>
wrote:

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> Hi Dale,
>
> We regularly do 5 color staining with Brilliant Violet 421, Brilliant
> Violet 510, AF488, AF594 (or AF555 if you choose) and AF647 or AF633 with a
> widefield scope and some minor filter changes.  A lot of microscopists
> still don't know about the Brilliant Violet polymeric fluors since most
> people are using them only in flow cytometry.  But, BV421 has an EC of 2.5
> million and a QY of 65% in PBS with 450nm/450nm ex/em.  You can see a 5
> color spleen with our normal Olympus IX83.  Though, we use direct
> conjugates for everything we can to overcome the species-dependent
> secondary detection issues you encounter with multiplexing microscopy.
>
> Outside of that, if you want to strip fluorescence, the only way to
> consistently kill any fluorophore is oxidation.  A planar fluor must remain
> rigid to resonate energy.  That's why some people will use peroxide
> incubations, to break a bond, release the rigidity of the hydrocarbon.
> Problem with any source of oxidation that can break a fluorophore, it'll
> also hurt cellular integrity/structure for the same reason, it's lack of
> selectivity.  You have probably heard of people quenching endogenous
> fluorescence this same way.
>
> Just my two cents!
> Kelly
>
>
> Kelly Lundsten
> Business Segment Manager, Advanced Cytometry
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> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]]
> On Behalf Of Dale Moulding
> Sent: Thursday, February 16, 2017 8:13 AM
> To: [log in to unmask]
> Subject: destroy alexa fluor fluorescence to allow restaining
>
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> Dear all,
> does anyone know of a way to irreversibly destroy alexa fluor fluorescence
> so a sample can be stained with a second set of dyes?
> We do whole mount staining for 3 antigens with Alexa 488, 568 and 633. We
> would then like to re-image the same samples with cellmask and phalloidin.
> Is there any way to kill alexa fluor fluorescence without destroying
> membranes and phalloidin binding?
> Cheers
> Dale
>