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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:[log in to unmask]] On Behalf Of Kelly Lundsten
Sent: Thursday, February 16, 2017 4:59 PM
To: [log in to unmask]
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: [log in to unmask]
Telephone: 773.633.4774
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:[log in to unmask]] On Behalf Of [log in to unmask]
Sent: Thursday, February 16, 2017 1:35 PM
To: [log in to unmask]
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 [log in to unmask]
On 17/2/17, 4:54 am, "Confocal Microscopy List on behalf of Kelly Lundsten" <[log in to unmask] on behalf of [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
Email: [log in to unmask]
Telephone: 773.633.4774
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:[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
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