destroy alexa fluor fluorescence to allow restaining

[Moulding, Dale]

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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

[Steffen Dietzel]

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If you can use blue cellmask and Sir-700-Actin instead of phalloidin,
you wouldn't have to. Else, instead of using A568 you could try with
A555 (or Cy3) and A594 (or Cy3.5 or Texas Red) and maybe A647 (or Cy5)
instead of A633. With 5 targets, I would go for a 5 color sample rather
than relabeling.

Steffen


Am 16.02.2017 um 17:12 schrieb Dale Moulding:

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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
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Biomedical Center (BMC)
Head of the Core Facility Bioimaging

Großhaderner Straße 9
D-82152 Planegg-Martinsried
Germany

http://www.bioimaging.bmc.med.uni-muenchen.de

[Martin Wessendorf-2]

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Dear Dale--

I know of no way to selectively quench the fluorescence of Alexa
fluorochromes.  However, back in the 1980s, we'd occasionally use
elution-restaining to multi-stain tissue if we needed to localize two
different antigens with primary antibodies raised in the same species.  
It involves treating the sample with potassium permanganate and
sulphuric acid--not gentle, and background goes up.  If you're stuck, it
might provide an approach.  A number of authors have used it to elute
fluorescently labeled antibodies.

Here's the reference:
Tramu,  G.,  A.  Pillez,  and  J.  Leonardelli  (1978)  An efficient  
method of  antibody  elution  for  the  successive or  simultaneous  
location  of two
antigens  by  immunocytochemistry.  J.  Histochem.  Cytochem. 26:  322-324.

Good luck!

Martin




On 2/16/2017 10:12 AM, Dale Moulding wrote:

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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

--
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Assoc Prof, Dept Neuroscience                 lab: [phone]
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[George McNamara]

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Hi Dale,

These are chemically different classes, so do not count on one method
working for all.

While not specifically mentioned by you, Alexa Fluor 647 is a Cy5
analog, should be destroyed by the Gerdes et al (GE multiOmyx) method, see

Highly multiplexed single-cell analysis of formalin-fixed,
paraffin-embedded cancer tissue.
Gerdes MJ, Sevinsky CJ, Sood A, Adak S, Bello MO, Bordwell A, Can A,
Corwin A, Dinn S, Filkins RJ, Hollman D, Kamath V, Kaanumalle S, Kenny
K, Larsen M, Lazare M, Li Q, Lowes C, McCulloch CC, McDonough E,
Montalto MC, Pang Z, Rittscher J, Santamaria-Pang A, Sarachan BD, Seel
ML, Seppo A, Shaikh K, Sui Y, Zhang J, Ginty F.
Proc Natl Acad Sci U S A. 2013 Jul 16;110(29):11982-7. doi:
10.1073/pnas.1300136110.
PMID: 23818604

and Gerdes patent.

May be simpler to just strip the antibodies off (be sure to wear PPE -
personal protective equipment -- while stripping antibodies).

See slides 11 and 12 of

https://digitalpathologyassociation.org/_data/files/2014_Pathology_Visions/PV14_Presentations/11_Day_2_Opening_Presentation_Levenson.pdf

for a first round 10plex. Also mentioned at

https://www.cacds.uh.edu/research/domain-applications/mapping-brain-tissue-alterations/

enjoy,

George

p.s. improving immunofluorescence on FFPE tissue sections - I encourage
checking out

Formaldehyde scavengers function as novel antigen retrieval agents.
Vollert CT, Moree WJ, Gregory S, Bark SJ, Eriksen JL.
Sci Rep. 2015 Nov 27;5:17322. doi: 10.1038/srep17322.
PMID: 26612041

and their corresponding patent (US 9506928,
http://www.freepatentsonline.com/9506928.html ), and to keep an eye on
the company, www.teomics.com, for product(s). I do not have a financial
connection to them (I met Craig V at  JLabs@TMC events recently). They
do not present quantitation on how much these 'formaldehyde scavengers'
improve FFPE tissue sections antigen retrieval by -- my hope is 20x
improement for 80% of epitopes ... which I think would greatly improve
direct immunofluorescence, which in turn would enable multiplexing. Of
course I had high hopes for an A.R. method a decade ago (PubMed
20025485, 15637333) which did not catch on.


On 2/16/2017 10:12 AM, Dale Moulding wrote:

> *****
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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

--


George McNamara, PhD
Houston, TX 77054
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75/
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650

[Kelly Lundsten-2]

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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
Email: [hidden email]
Telephone: [phone]
Website: www.biolegend.com
BioLegend
9727 Pacific Heights Blvd., San Diego, CA 92121, USA

This message (including any attachments) may contain confidential, proprietary, privileged and/or private information. The information is intended to be for the use of the individual or entity designated above. If you are not the intended recipient of this message, please notify the sender immediately, and delete the message any attachments. Any disclosure, distribution or other use of this message or any attachments by an individual or entity other than the intended recipient is prohibited.



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Dale Moulding
Sent: Thursday, February 16, 2017 8:13 AM
To: [hidden email]
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

[Romain Laine]

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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: ([phone]




On Thu, Feb 16, 2017 at 5:54 PM, Kelly Lundsten <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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
> Email: [hidden email]
> Telephone: [phone]
> Website: www.biolegend.com
> BioLegend
> 9727 Pacific Heights Blvd., San Diego, CA 92121, USA
>
> This message (including any attachments) may contain confidential,
> proprietary, privileged and/or private information. The information is
> intended to be for the use of the individual or entity designated above. If
> you are not the intended recipient of this message, please notify the
> sender immediately, and delete the message any attachments. Any disclosure,
> distribution or other use of this message or any attachments by an
> individual or entity other than the intended recipient is prohibited.
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Dale Moulding
> Sent: Thursday, February 16, 2017 8:13 AM
> To: [hidden email]
> Subject: destroy alexa fluor fluorescence to allow restaining
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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
>

[Rosemary.White]

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Hi Kelly,

Do you have any references detailing this method? This would be useful for us plant people as well, who have to deal with chloroplasts and other autofluorescence.

thanks much,
Rosemary

Dr Rosemary White
CSIRO Black Mountain
GPO Box 1700
Canberra, ACT 2601
Australia
Adjunct Prof, EH Graham Centre, CSU
& Research School of Biology, ANU
 
T 61 2 6246 5475
E [hidden email]


On 17/2/17, 4:54 am, "Confocal Microscopy List on behalf of Kelly Lundsten" <[hidden email] on behalf of [hidden email]> wrote:

    *****
    To join, leave or search the confocal microscopy listserv, go to:
    http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
    Post images on http://www.imgur.com and include the link in your posting.
    *****
   
    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
    Email: [hidden email]
    Telephone: [phone]
    Website: www.biolegend.com
    BioLegend
    9727 Pacific Heights Blvd., San Diego, CA 92121, USA
   
    This message (including any attachments) may contain confidential, proprietary, privileged and/or private information. The information is intended to be for the use of the individual or entity designated above. If you are not the intended recipient of this message, please notify the sender immediately, and delete the message any attachments. Any disclosure, distribution or other use of this message or any attachments by an individual or entity other than the intended recipient is prohibited.
   
   
   
    -----Original Message-----
    From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Dale Moulding
    Sent: Thursday, February 16, 2017 8:13 AM
    To: [hidden email]
    Subject: destroy alexa fluor fluorescence to allow restaining
   
    *****
    To join, leave or search the confocal microscopy listserv, go to:
    http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
    Post images on http://www.imgur.com and include the link in your posting.
    *****
   
    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
   

[Kelly Lundsten-2]

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Hi Rosemary,

I totally concur on the sodium borohydride as well though I've never done that one myself.  The peroxide is usually like 10-15% done on IHC-paraffin embedded tissue partly to reduce the autofluorescence but also to quench the endogenous peroxidase for people using HRP conjugates on the tissue later, so two birds one stone from the IHC perspective.  I don't use the method myself so I was just googling for a protocol.  

I agree with Jason as well.  All three of these methods H2O2, sodium borohydride and UV illumination or white light sources are all methods that Molecular Probes would recommend back in the day.  UV and white light excitation are causing ROS production as the fluor excites and relaxes in the water environment, ROS are produced that go back to break the double bonds of the benzenes/hydrocarbons of the organic fluor structure.  I don't know exactly how sodium borohydride does it though, since it's not an oxidizing agent. I suspect it quenches the fluors by making them no longer quantum efficient through a different reducing mechanism.  Maybe Jason knows this one.  Sorry I don't have much time to look into this more but here is a link to Abcam's blurb on it:  http://www.abcam.com/kits/blocking-for-ihc .  I remember the key always either being reduce the % H2O2 and lengthen the time or increase the %H2O2 to reduce the time.... all an optimization for the tissue and intensity of the signal you are hoping to destroy while doing minimal damage to the tissue.

Good luck all!
Kelly Lundsten
Business Segment Manager, Advanced Cytometry
Email: [hidden email]
Telephone: [phone]
Website: www.biolegend.com
BioLegend
9727 Pacific Heights Blvd., San Diego, CA 92121, USA

This message (including any attachments) may contain confidential, proprietary, privileged and/or private information. The information is intended to be for the use of the individual or entity designated above. If you are not the intended recipient of this message, please notify the sender immediately, and delete the message any attachments. Any disclosure, distribution or other use of this message or any attachments by an individual or entity other than the intended recipient is prohibited.




-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of [hidden email]
Sent: Thursday, February 16, 2017 1:35 PM
To: [hidden email]
Subject: Re: destroy alexa fluor fluorescence to allow restaining

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Hi Kelly,

Do you have any references detailing this method? This would be useful for us plant people as well, who have to deal with chloroplasts and other autofluorescence.

thanks much,
Rosemary

Dr Rosemary White
CSIRO Black Mountain
GPO Box 1700
Canberra, ACT 2601
Australia
Adjunct Prof, EH Graham Centre, CSU
& Research School of Biology, ANU
 
T 61 2 6246 5475
E [hidden email]


On 17/2/17, 4:54 am, "Confocal Microscopy List on behalf of Kelly Lundsten" <[hidden email] on behalf of [hidden email]> wrote:

    *****
    To join, leave or search the confocal microscopy listserv, go to:
    http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
    Post images on http://www.imgur.com and include the link in your posting.
    *****
   
    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
    Email: [hidden email]
    Telephone: [phone]
    Website: www.biolegend.com
    BioLegend
    9727 Pacific Heights Blvd., San Diego, CA 92121, USA
   
    This message (including any attachments) may contain confidential, proprietary, privileged and/or private information. The information is intended to be for the use of the individual or entity designated above. If you are not the intended recipient of this message, please notify the sender immediately, and delete the message any attachments. Any disclosure, distribution or other use of this message or any attachments by an individual or entity other than the intended recipient is prohibited.
   
   
   
    -----Original Message-----
    From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Dale Moulding
    Sent: Thursday, February 16, 2017 8:13 AM
    To: [hidden email]
    Subject: destroy alexa fluor fluorescence to allow restaining
   
    *****
    To join, leave or search the confocal microscopy listserv, go to:
    http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
    Post images on http://www.imgur.com and include the link in your posting.
    *****
   
    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
   

[Jeffrey Carmichael]

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COMMERCIAL RESPONSE

Hi Dale,

I agree with Kelly that you can do this with 5 colors relatively
easily *(although
commercial responses should be labeled as such).*

Below is a link to a slide deck on our website outlining the imaging scheme
and filter sets to best separate these.  BD Biosciences also offers the
BV480 (like Aqua or CFP, but much, much brighter) which allows for distinct
excitation and emission ranges for all 5 fluors.

https://www.chroma.com/sites/ <goog_1687719810>default/files/5-CHANNEL%
<goog_1687719810>20FLUORESCENCE%20IMAGING% <goog_1687719810>
20SIMPLIFIED%20-%20Reliable% <goog_1687719810>20Multiplexing%20for%20the%
<goog_1687719810>20Non-Specialist_New.pdf

Chroma does not profit from sales of any of the fluorophores, only from the
filters.  We also offer two different 5-band filter sets to accommodate
this:

https://www.chroma.com/products/sets/89903-et-bv421-bv480-af488-af568-af647-quinta-band-set
https://www.chroma.com/products/sets/89904-et-405-445-514-561-640nm-laser-quinta-band-set

I was initially skeptical that we could achieve good separation between
BV480 and AF488, but with the right filter sets (specified in the slide
deck) you can indeed.

Good luck,
Jeff



*Jeff Carmichael*

*Technical and Product Marketing Manager*

*[hidden email] <[hidden email]>*Chroma Technology Corp.

*an employee owned company*
*10 Imtec Lane*
*Bellows Falls, VT  05301*
*802-428-2528 Office*
*802-428-2528 Fax**800-824-7662 Toll Free*

On Thu, Feb 16, 2017 at 11:12 AM, Dale Moulding <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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
>

[mcammer]

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From a protocol we use for bleaching CNS before staining, maybe would work for stripping stains too:

Incubate with NaIO4 (21.4 mg/ml in PBS) 15 min. followed by PBS wash
Alternative bleaching solutions can be fresh NaBH4 or glycine followed by PBS wash


=*===========================================================*=
 Michael Cammer, DART Microscopy Laboratory, NYU Langone Medical Center
    Cell:  914-309-3270     Office: Skirball 2nd Floor main office, back right
      http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Kelly Lundsten
Sent: Thursday, February 16, 2017 4:59 PM
To: [hidden email]
Subject: Re: destroy alexa fluor fluorescence to allow restaining

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Hi Rosemary,

I totally concur on the sodium borohydride as well though I've never done that one myself.  The peroxide is usually like 10-15% done on IHC-paraffin embedded tissue partly to reduce the autofluorescence but also to quench the endogenous peroxidase for people using HRP conjugates on the tissue later, so two birds one stone from the IHC perspective.  I don't use the method myself so I was just googling for a protocol.  

I agree with Jason as well.  All three of these methods H2O2, sodium borohydride and UV illumination or white light sources are all methods that Molecular Probes would recommend back in the day.  UV and white light excitation are causing ROS production as the fluor excites and relaxes in the water environment, ROS are produced that go back to break the double bonds of the benzenes/hydrocarbons of the organic fluor structure.  I don't know exactly how sodium borohydride does it though, since it's not an oxidizing agent. I suspect it quenches the fluors by making them no longer quantum efficient through a different reducing mechanism.  Maybe Jason knows this one.  Sorry I don't have much time to look into this more but here is a link to Abcam's blurb on it:  https://urldefense.proofpoint.com/v2/url?u=http-3A__www.abcam.com_kits_blocking-2Dfor-2Dihc&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=tTSr0amUjZOeOx4X_0vQqaRXof4boefIoOxcusaWDdI&s=-oddrxaWstgi44kuXoZsDaU6GeZ1YB4nJW8600DXdEk&e=  .  I remember the key always either being reduce the % H2O2 and lengthen the time or increase the %H2O2 to reduce the time.... all an optimization for the tissue and intensity of the signal you are hoping to destroy while doing minimal damage to the tissue.

Good luck all!
Kelly Lundsten
Business Segment Manager, Advanced Cytometry
Email: [hidden email]
Telephone: [phone]
Website: www.biolegend.com
BioLegend
9727 Pacific Heights Blvd., San Diego, CA 92121, USA

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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of [hidden email]
Sent: Thursday, February 16, 2017 1:35 PM
To: [hidden email]
Subject: Re: destroy alexa fluor fluorescence to allow restaining

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Hi Kelly,

Do you have any references detailing this method? This would be useful for us plant people as well, who have to deal with chloroplasts and other autofluorescence.

thanks much,
Rosemary

Dr Rosemary White
CSIRO Black Mountain
GPO Box 1700
Canberra, ACT 2601
Australia
Adjunct Prof, EH Graham Centre, CSU
& Research School of Biology, ANU
 
T 61 2 6246 5475
E [hidden email]


On 17/2/17, 4:54 am, "Confocal Microscopy List on behalf of Kelly Lundsten" <[hidden email] on behalf of [hidden email]> wrote:

    *****
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    Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DQIFaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=tTSr0amUjZOeOx4X_0vQqaRXof4boefIoOxcusaWDdI&s=jaSbIuPJZfYtZh5pwdlcbgJGVosDcO0_U6mn4nNKRFw&e=  and include the link in your posting.
    *****
   
    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
    Email: [hidden email]
    Telephone: [phone]
    Website: www.biolegend.com
    BioLegend
    9727 Pacific Heights Blvd., San Diego, CA 92121, USA
   
    This message (including any attachments) may contain confidential, proprietary, privileged and/or private information. The information is intended to be for the use of the individual or entity designated above. If you are not the intended recipient of this message, please notify the sender immediately, and delete the message any attachments. Any disclosure, distribution or other use of this message or any attachments by an individual or entity other than the intended recipient is prohibited.
   
   
   
    -----Original Message-----
    From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Dale Moulding
    Sent: Thursday, February 16, 2017 8:13 AM
    To: [hidden email]
    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
   


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[Jeffrey Carmichael]

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COMMERCIAL RESPONSE

Dale,

In case the link didn't go through properly, I'll try again:

https://www.chroma.com/sites/default/files/5-CHANNEL%20FLUORESCENCE%20IMAGING%20SIMPLIFIED%20-%20Reliable%20Multiplexing%20for%20the%20Non-Specialist_New.pdf

Jeff


*Jeff Carmichael*

*Technical and Product Marketing Manager*

*[hidden email] <[hidden email]>*Chroma Technology Corp.

*an employee owned company*
*10 Imtec Lane*
*Bellows Falls, VT  05301*
*802-428-2528 Office*
*802-428-2528 Fax**800-824-7662 Toll Free*

On Thu, Feb 16, 2017 at 11:12 AM, Dale Moulding <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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
>

[Fyl Tafas]

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PW CHANGE MICROSPIM1_

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jeffrey Carmichael
Sent: Thursday, February 16, 2017 5:57 PM
To: [hidden email]
Subject: Re: destroy alexa fluor fluorescence to allow restaining

*****
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COMMERCIAL RESPONSE

Dale,

In case the link didn't go through properly, I'll try again:

https://www.chroma.com/sites/default/files/5-CHANNEL%20FLUORESCENCE%20IMAGING%20SIMPLIFIED%20-%20Reliable%20Multiplexing%20for%20the%20Non-Specialist_New.pdf

Jeff


*Jeff Carmichael*

*Technical and Product Marketing Manager*

*[hidden email] <[hidden email]>*Chroma Technology Corp.

*an employee owned company*
*10 Imtec Lane*
*Bellows Falls, VT  05301*
*802-428-2528 Office*
*802-428-2528 Fax**800-824-7662 Toll Free*

On Thu, Feb 16, 2017 at 11:12 AM, Dale Moulding <[hidden email]>
wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> 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
>

[Moulding, Dale]

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Dear all,
wow, thanks for the extremely helpful replies. I neglected to mention we have Dapi in the mix, so are looking for 6 colour imaging. From the replies this looks achievable.
We’re not committed to any specific fluorophores, so it seems simply using Cy3 & Cy5 will allow us to re-use those channels for the Cell mask and Phalloidin.
I’d not considered the BV fluorophores, these certainly look useful, as are the various chemicals (NaBH4, NaIO4, Glycine!?!) so we have plenty of options.
Thanks!
Dale

[Jim Mansfield]

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If you are just looking for a 5-plex, 6-color, staining, there are lots of publications about how to do that without having to bleach, restain, reimage and then deal with all of the imaging artifacts introduced!

https://jitc.biomedcentral.com/articles/10.1186/s40425-015-0091-z

http://www.sciencedirect.com/science/article/pii/S1046202314002837

http://www.nature.com/articles/srep09534?WT.feed_name=subjects_imaging-the-immune-system

http://link.springer.com/protocol/10.1007/978-1-4939-6730-8_5

Plus, there are commercial systems which enable this kind of staining using autostainers. Much of the above is possible on Leica Bond RX stainers, and Ventana/Roche has recently announced a 5-plex assay on their stainers.

The publications above have all been optimized for thin FFPE tissue sections, so they might have to be modified for your samples.

Best,

Jim

(no commercial interest)


Jim Mansfield
[hidden email]
[phone]

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Dale Moulding
Sent: February 20, 2017 7:56 AM
To: [hidden email]
Subject: Re: destroy alexa fluor fluorescence to allow restaining

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Dear all,
wow, thanks for the extremely helpful replies. I neglected to mention we have Dapi in the mix, so are looking for 6 colour imaging. From the replies this looks achievable.
We’re not committed to any specific fluorophores, so it seems simply using Cy3 & Cy5 will allow us to re-use those channels for the Cell mask and Phalloidin.
I’d not considered the BV fluorophores, these certainly look useful, as are the various chemicals (NaBH4, NaIO4, Glycine!?!) so we have plenty of options.
Thanks!
Dale