mounting media and anti-fade reagents

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Knecht, David Knecht, David
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mounting media and anti-fade reagents

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I recently switched from fluorescein/rhodamine labels to Alexa 477/568.  Much to my surprise, the alexa dyes bleached very rapidly in a spinning disk microscope with just buffer as mounting media.  I then added my usual phenylene diamine/glycerol based mounting media and the fluorescence disappeared completely for both alexa dyes.  I found this problem well known in the literature.  So what do people used for home made mounting media cocktails for alexa dyes?  Alternatively, are there any dyes that work well with PDA?  I am tempted to just switch back to fluorescein and rhodamine.  Thanks- Dave

Dr. David Knecht
Professor , Department of Molecular and Cell Biology
University of Connecticut
91 N. Eagleville Rd.
U-3125
Storrs, CT 06269-3125
860-486-2200
Indig, Fred (NIH/NIA/IRP) [E] Indig, Fred (NIH/NIA/IRP) [E]
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Re: mounting media and anti-fade reagents

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Dear David,


Try DABCO for home-made, I can provide a recipe if you wish.


Among commercial anti-fade mounting media, we found that Prolong Gold/Diamond works best for our applications

in both confocal and iSIM (not so different from your spinning disc).


Best Wishes,

Fred


Fred E. Indig, Ph.D.

Head, Confocal Imaging Facility


Biomedical Research Center (BRC) Rm 8B135

National Institute on Aging/NIH

251 Bayview Blvd.

Baltimore, MD  21224-6825


Tel. 410-558-8173

Fax  410-558-8236

[hidden email]<mailto:[hidden email]>


________________________________
From: Knecht, David <[hidden email]>
Sent: Wednesday, March 28, 2018 10:53:13 AM
To: [hidden email]
Subject: mounting media and anti-fade reagents

*****
To join, leave or search the confocal microscopy listserv, go to:
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Post images on http://www.imgur.com and include the link in your posting.
*****

I recently switched from fluorescein/rhodamine labels to Alexa 477/568.  Much to my surprise, the alexa dyes bleached very rapidly in a spinning disk microscope with just buffer as mounting media.  I then added my usual phenylene diamine/glycerol based mounting media and the fluorescence disappeared completely for both alexa dyes.  I found this problem well known in the literature.  So what do people used for home made mounting media cocktails for alexa dyes?  Alternatively, are there any dyes that work well with PDA?  I am tempted to just switch back to fluorescein and rhodamine.  Thanks- Dave

Dr. David Knecht
Professor , Department of Molecular and Cell Biology
University of Connecticut
91 N. Eagleville Rd.
U-3125
Storrs, CT 06269-3125
860-486-2200
Tony Kowal Tony Kowal
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Re: mounting media and anti-fade reagents

In reply to this post by Knecht, David
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Hi Dave -

We use alexa fluor dyes almost exclusively in our lab, and we mount in Mowial or ProLong Diamond.  For confocal, I think that the fluorescence is more stable with the ProLong Diamond.

Hope that helps -

Tony
Alison J. North Alison J. North
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Re: mounting media and anti-fade reagents

In reply to this post by Indig, Fred (NIH/NIA/IRP) [E]
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Hi David and Fred,

I too am a fan of Prolong Diamond.  It works better than Prolong Gold on
blue-emitting dyes and also on the fluorescent proteins. We do not allow
it to cure, otherwise it squishes everything flat (as does Mowiol) which
is obviously no good for 3D-imaging.  But if you just wick away the
excess for a minute and then seal immediately around the edge of the
coverslip, it works really well on all of our microscopes.

For some other dyes (non-Alexa), like the Bodipy dyes and also some blue
dyes like DyLight 405, I find that home-made mountants containing
N-propyl gallate work best.  It all depends on the exact dye - for
example, Vectashield kills Alexa 647, while DABCO kills Alexa 488 - they
knock the initial intensities down by about 75%, according to a graph I
got from Molecular Probes, and we've certainly seen that in practice for
Vectashield and AF647.  I believe that Vectashield contains Phenylene
Diamene (though they won't tell you), which is great for FITC but
terrible for far red dyes, nor so good for some of the red Alexa dyes
e.g. 594.

Best,

Alison


On 3/28/2018 11:18 AM, Indig, Fred (NIH/NIA/IRP) [E] wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFAg&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=_intRa89BmLCxHGeG_MoI8OfuqtSxU79ebDpHBN5Dgk&s=Am7uR6ZnYMVB27ja-oDtGIdyuuTkMVpwaM9JphaaxVk&e=
> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFAg&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=_intRa89BmLCxHGeG_MoI8OfuqtSxU79ebDpHBN5Dgk&s=ngLTCD5oicUBC3RCSYKKi2eG5hQ9tvm0yD_v6bD7cWw&e= and include the link in your posting.
> *****
>
> Dear David,
>
>
> Try DABCO for home-made, I can provide a recipe if you wish.
>
>
> Among commercial anti-fade mounting media, we found that Prolong Gold/Diamond works best for our applications
>
> in both confocal and iSIM (not so different from your spinning disc).
>
>
> Best Wishes,
>
> Fred
>
>
> Fred E. Indig, Ph.D.
>
> Head, Confocal Imaging Facility
>
>
> Biomedical Research Center (BRC) Rm 8B135
>
> National Institute on Aging/NIH
>
> 251 Bayview Blvd.
>
> Baltimore, MD  21224-6825
>
>
> Tel. 410-558-8173
>
> Fax  410-558-8236
>
> [hidden email]<mailto:[hidden email]>
>
>
> ________________________________
> From: Knecht, David <[hidden email]>
> Sent: Wednesday, March 28, 2018 10:53:13 AM
> To: [hidden email]
> Subject: mounting media and anti-fade reagents
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFAg&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=_intRa89BmLCxHGeG_MoI8OfuqtSxU79ebDpHBN5Dgk&s=Am7uR6ZnYMVB27ja-oDtGIdyuuTkMVpwaM9JphaaxVk&e=
> Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFAg&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=_intRa89BmLCxHGeG_MoI8OfuqtSxU79ebDpHBN5Dgk&s=ngLTCD5oicUBC3RCSYKKi2eG5hQ9tvm0yD_v6bD7cWw&e= and include the link in your posting.
> *****
>
> I recently switched from fluorescein/rhodamine labels to Alexa 477/568.  Much to my surprise, the alexa dyes bleached very rapidly in a spinning disk microscope with just buffer as mounting media.  I then added my usual phenylene diamine/glycerol based mounting media and the fluorescence disappeared completely for both alexa dyes.  I found this problem well known in the literature.  So what do people used for home made mounting media cocktails for alexa dyes?  Alternatively, are there any dyes that work well with PDA?  I am tempted to just switch back to fluorescein and rhodamine.  Thanks- Dave
>
> Dr. David Knecht
> Professor , Department of Molecular and Cell Biology
> University of Connecticut
> 91 N. Eagleville Rd.
> U-3125
> Storrs, CT 06269-3125
> 860-486-2200

--
Alison J. North, Ph.D.,
Research Associate Professor and
Senior Director of the Bio-Imaging Resource Center,
The Rockefeller University,
1230 York Avenue,
New York,
NY 10065.
Tel: office ++ 212 327 7488
Tel: lab     ++ 212 327 7486
Fax:         ++ 212 327 7489
Carol J. Bayles Carol J. Bayles
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Re: mounting media and anti-fade reagents

*****
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.
*****

I am having a problem mounting cells with MitoTracker Red.  I am using the fixable version, and they look beautiful after fixing.  But if I add Prolong Diamond or Vectashield, after curing all the MitoTracker is in the nucleus and with high background in the cytoplasm.  Anyone know how to mount a slide with MitoTracker?  I just want to have pretty slides, my cells have GFP-tubulin also.  The microtubules look fine.
Thanks,
Carol
<><><><><><><><>
Carol Bayles
BRC-Imaging-FACS
B46 Weill Hall
Cornell University
Ithaca, NY 14853



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Alison North
Sent: Wednesday, March 28, 2018 11:43 AM
To: [hidden email]
Subject: Re: mounting media and anti-fade reagents

*****
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 David and Fred,

I too am a fan of Prolong Diamond.  It works better than Prolong Gold on blue-emitting dyes and also on the fluorescent proteins. We do not allow it to cure, otherwise it squishes everything flat (as does Mowiol) which is obviously no good for 3D-imaging.  But if you just wick away the excess for a minute and then seal immediately around the edge of the coverslip, it works really well on all of our microscopes.

For some other dyes (non-Alexa), like the Bodipy dyes and also some blue dyes like DyLight 405, I find that home-made mountants containing N-propyl gallate work best.  It all depends on the exact dye - for example, Vectashield kills Alexa 647, while DABCO kills Alexa 488 - they knock the initial intensities down by about 75%, according to a graph I got from Molecular Probes, and we've certainly seen that in practice for Vectashield and AF647.  I believe that Vectashield contains Phenylene Diamene (though they won't tell you), which is great for FITC but terrible for far red dyes, nor so good for some of the red Alexa dyes e.g. 594.

Best,

Alison


On 3/28/2018 11:18 AM, Indig, Fred (NIH/NIA/IRP) [E] wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-
> 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFAg&c=JeTkUgVztGMmhKYjxsy2rfoW
> YibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=_i
> ntRa89BmLCxHGeG_MoI8OfuqtSxU79ebDpHBN5Dgk&s=Am7uR6ZnYMVB27ja-oDtGIdyuu
> TkMVpwaM9JphaaxVk&e= Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFAg&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=_intRa89BmLCxHGeG_MoI8OfuqtSxU79ebDpHBN5Dgk&s=ngLTCD5oicUBC3RCSYKKi2eG5hQ9tvm0yD_v6bD7cWw&e= and include the link in your posting.
> *****
>
> Dear David,
>
>
> Try DABCO for home-made, I can provide a recipe if you wish.
>
>
> Among commercial anti-fade mounting media, we found that Prolong
> Gold/Diamond works best for our applications
>
> in both confocal and iSIM (not so different from your spinning disc).
>
>
> Best Wishes,
>
> Fred
>
>
> Fred E. Indig, Ph.D.
>
> Head, Confocal Imaging Facility
>
>
> Biomedical Research Center (BRC) Rm 8B135
>
> National Institute on Aging/NIH
>
> 251 Bayview Blvd.
>
> Baltimore, MD  21224-6825
>
>
> Tel. 410-558-8173
>
> Fax  410-558-8236
>
> [hidden email]<mailto:[hidden email]>
>
>
> ________________________________
> From: Knecht, David <[hidden email]>
> Sent: Wednesday, March 28, 2018 10:53:13 AM
> To: [hidden email]
> Subject: mounting media and anti-fade reagents
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-
> 2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFAg&c=JeTkUgVztGMmhKYjxsy2rfoW
> YibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=_i
> ntRa89BmLCxHGeG_MoI8OfuqtSxU79ebDpHBN5Dgk&s=Am7uR6ZnYMVB27ja-oDtGIdyuu
> TkMVpwaM9JphaaxVk&e= Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFAg&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=_intRa89BmLCxHGeG_MoI8OfuqtSxU79ebDpHBN5Dgk&s=ngLTCD5oicUBC3RCSYKKi2eG5hQ9tvm0yD_v6bD7cWw&e= and include the link in your posting.
> *****
>
> I recently switched from fluorescein/rhodamine labels to Alexa
> 477/568.  Much to my surprise, the alexa dyes bleached very rapidly in
> a spinning disk microscope with just buffer as mounting media.  I then
> added my usual phenylene diamine/glycerol based mounting media and the
> fluorescence disappeared completely for both alexa dyes.  I found this
> problem well known in the literature.  So what do people used for home
> made mounting media cocktails for alexa dyes?  Alternatively, are
> there any dyes that work well with PDA?  I am tempted to just switch
> back to fluorescein and rhodamine.  Thanks- Dave
>
> Dr. David Knecht
> Professor , Department of Molecular and Cell Biology University of
> Connecticut
> 91 N. Eagleville Rd.
> U-3125
> Storrs, CT 06269-3125
> 860-486-2200

--
Alison J. North, Ph.D.,
Research Associate Professor and
Senior Director of the Bio-Imaging Resource Center, The Rockefeller University,
1230 York Avenue,
New York,
NY 10065.
Tel: office ++ 212 327 7488
Tel: lab     ++ 212 327 7486
Fax:         ++ 212 327 7489
Knecht, David Knecht, David
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Re: mounting media and anti-fade reagents

In reply to this post by Indig, Fred (NIH/NIA/IRP) [E]
*****
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.
*****

I am trying to avoid the commercial preparations as they are quite expensive for the volumes we are using in a classroom setting.  I did use our DABCO based mounting media for the Alexas and it was an improvement.  I know that PPD for FITC/TRITC  was cheap and by far the best antifade I tried, and never had a problem with background- but I did not use it in the 405 channel.  Dave

Dr. David Knecht
Professor , Department of Molecular and Cell Biology
University of Connecticut
91 N. Eagleville Rd.
U-3125
Storrs, CT 06269-3125
860-486-2200

> On Mar 28, 2018, at 11:18 AM, Indig, Fred (NIH/NIA/IRP) [E] <[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 David,
>
>
> Try DABCO for home-made, I can provide a recipe if you wish.
>
>
> Among commercial anti-fade mounting media, we found that Prolong Gold/Diamond works best for our applications
>
> in both confocal and iSIM (not so different from your spinning disc).
>
>
> Best Wishes,
>
> Fred
>
>
> Fred E. Indig, Ph.D.
>
> Head, Confocal Imaging Facility
>
>
> Biomedical Research Center (BRC) Rm 8B135
>
> National Institute on Aging/NIH
>
> 251 Bayview Blvd.
>
> Baltimore, MD  21224-6825
>
>
> Tel. 410-558-8173
>
> Fax  410-558-8236
>
> [hidden email]<mailto:[hidden email]>
>
>
> ________________________________
> From: Knecht, David <[hidden email]>
> Sent: Wednesday, March 28, 2018 10:53:13 AM
> To: [hidden email]
> Subject: mounting media and anti-fade reagents
>
> *****
> 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.
> *****
>
> I recently switched from fluorescein/rhodamine labels to Alexa 477/568.  Much to my surprise, the alexa dyes bleached very rapidly in a spinning disk microscope with just buffer as mounting media.  I then added my usual phenylene diamine/glycerol based mounting media and the fluorescence disappeared completely for both alexa dyes.  I found this problem well known in the literature.  So what do people used for home made mounting media cocktails for alexa dyes?  Alternatively, are there any dyes that work well with PDA?  I am tempted to just switch back to fluorescein and rhodamine.  Thanks- Dave
>
> Dr. David Knecht
> Professor , Department of Molecular and Cell Biology
> University of Connecticut
> 91 N. Eagleville Rd.
> U-3125
> Storrs, CT 06269-3125
> 860-486-2200
Douglas Richardson Douglas Richardson
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Re: mounting media and anti-fade reagents

In reply to this post by Alison J. North
*****
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*****

I've just recently switched to ProLong Glass, especially for thicker samples, SIM, and other super-resolution applications.  The improved RI match really helps when using oil immersion objectives.

-Doug

On Wed, Mar 28, 2018 at 11:42 AM, Alison North <[hidden email]<mailto:[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 David and Fred,

I too am a fan of Prolong Diamond.  It works better than Prolong Gold on blue-emitting dyes and also on the fluorescent proteins. We do not allow it to cure, otherwise it squishes everything flat (as does Mowiol) which is obviously no good for 3D-imaging.  But if you just wick away the excess for a minute and then seal immediately around the edge of the coverslip, it works really well on all of our microscopes.

For some other dyes (non-Alexa), like the Bodipy dyes and also some blue dyes like DyLight 405, I find that home-made mountants containing N-propyl gallate work best.  It all depends on the exact dye - for example, Vectashield kills Alexa 647, while DABCO kills Alexa 488 - they knock the initial intensities down by about 75%, according to a graph I got from Molecular Probes, and we've certainly seen that in practice for Vectashield and AF647.  I believe that Vectashield contains Phenylene Diamene (though they won't tell you), which is great for FITC but terrible for far red dyes, nor so good for some of the red Alexa dyes e.g. 594.

Best,

Alison


On 3/28/2018 11:18 AM, Indig, Fred (NIH/NIA/IRP) [E] wrote:
*****
To join, leave or search the confocal microscopy listserv, go to:
https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFAg&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=_intRa89BmLCxHGeG_MoI8OfuqtSxU79ebDpHBN5Dgk&s=Am7uR6ZnYMVB27ja-oDtGIdyuuTkMVpwaM9JphaaxVk&e=
Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFAg&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=_intRa89BmLCxHGeG_MoI8OfuqtSxU79ebDpHBN5Dgk&s=ngLTCD5oicUBC3RCSYKKi2eG5hQ9tvm0yD_v6bD7cWw&e= and include the link in your posting.
*****

Dear David,


Try DABCO for home-made, I can provide a recipe if you wish.


Among commercial anti-fade mounting media, we found that Prolong Gold/Diamond works best for our applications

in both confocal and iSIM (not so different from your spinning disc).


Best Wishes,

Fred


Fred E. Indig, Ph.D.

Head, Confocal Imaging Facility


Biomedical Research Center (BRC) Rm 8B135

National Institute on Aging/NIH

251 Bayview Blvd.

Baltimore, MD  21224-6825


Tel. 410-558-8173<tel:410-558-8173>

Fax  410-558-8236<tel:410-558-8236>

[hidden email]<mailto:[hidden email]><mailto:[hidden email]<mailto:[hidden email]>>


________________________________
From: Knecht, David <[hidden email]<mailto:[hidden email]>>
Sent: Wednesday, March 28, 2018 10:53:13 AM
To: [hidden email]<mailto:[hidden email]>
Subject: mounting media and anti-fade reagents

*****
To join, leave or search the confocal microscopy listserv, go to:
https://urldefense.proofpoint.com/v2/url?u=http-3A__lists.umn.edu_cgi-2Dbin_wa-3FA0-3Dconfocalmicroscopy&d=DwIFAg&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=_intRa89BmLCxHGeG_MoI8OfuqtSxU79ebDpHBN5Dgk&s=Am7uR6ZnYMVB27ja-oDtGIdyuuTkMVpwaM9JphaaxVk&e=
Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFAg&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=_intRa89BmLCxHGeG_MoI8OfuqtSxU79ebDpHBN5Dgk&s=ngLTCD5oicUBC3RCSYKKi2eG5hQ9tvm0yD_v6bD7cWw&e= and include the link in your posting.
*****

I recently switched from fluorescein/rhodamine labels to Alexa 477/568.  Much to my surprise, the alexa dyes bleached very rapidly in a spinning disk microscope with just buffer as mounting media.  I then added my usual phenylene diamine/glycerol based mounting media and the fluorescence disappeared completely for both alexa dyes.  I found this problem well known in the literature.  So what do people used for home made mounting media cocktails for alexa dyes?  Alternatively, are there any dyes that work well with PDA?  I am tempted to just switch back to fluorescein and rhodamine.  Thanks- Dave

Dr. David Knecht
Professor , Department of Molecula<https://maps.google.com/?q=artment+of+Molecula&entry=gmail&source=g>r and Cell Biology
University of Connecticut
91 N. Eagleville Rd.
U-3125
Storrs, CT 06269-3125
860-486-2200<tel:860-486-2200>

--
Alison J. North, Ph.D.,
Research Associate Professor and
Senior Director of the Bio-Imaging Resource <https://maps.google.com/?q=Director+of+the+Bio-Imaging+Resource+&entry=gmail&source=g> Center,
The Rockefeller University,
1230 York Avenue,
New York,
NY 10065.
Tel: office     ++ 212 327 7488
Tel: lab        ++ 212 327 7486
Fax:            ++ 212 327 7489
Jeremy Adler-5 Jeremy Adler-5
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Re: mounting media and anti-fade reagents

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Citiflour
https://www.citifluor.com

have a large range different mounting media with different RIs, fluidity and antifade agents.

Their prices are quite modest.

- no commercial interest.

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Doug Richardson
Sent: 29 March 2018 01:53
To: [hidden email]
Subject: Re: mounting media and anti-fade reagents

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I've just recently switched to ProLong Glass, especially for thicker samples, SIM, and other super-resolution applications.  The improved RI match really helps when using oil immersion objectives.

-Doug

On Wed, Mar 28, 2018 at 11:42 AM, Alison North <[hidden email]<mailto:[hidden email]>> wrote:
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Hi David and Fred,

I too am a fan of Prolong Diamond.  It works better than Prolong Gold on blue-emitting dyes and also on the fluorescent proteins. We do not allow it to cure, otherwise it squishes everything flat (as does Mowiol) which is obviously no good for 3D-imaging.  But if you just wick away the excess for a minute and then seal immediately around the edge of the coverslip, it works really well on all of our microscopes.

For some other dyes (non-Alexa), like the Bodipy dyes and also some blue dyes like DyLight 405, I find that home-made mountants containing N-propyl gallate work best.  It all depends on the exact dye - for example, Vectashield kills Alexa 647, while DABCO kills Alexa 488 - they knock the initial intensities down by about 75%, according to a graph I got from Molecular Probes, and we've certainly seen that in practice for Vectashield and AF647.  I believe that Vectashield contains Phenylene Diamene (though they won't tell you), which is great for FITC but terrible for far red dyes, nor so good for some of the red Alexa dyes e.g. 594.

Best,

Alison


On 3/28/2018 11:18 AM, Indig, Fred (NIH/NIA/IRP) [E] wrote:
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Dear David,


Try DABCO for home-made, I can provide a recipe if you wish.


Among commercial anti-fade mounting media, we found that Prolong Gold/Diamond works best for our applications

in both confocal and iSIM (not so different from your spinning disc).


Best Wishes,

Fred


Fred E. Indig, Ph.D.

Head, Confocal Imaging Facility


Biomedical Research Center (BRC) Rm 8B135

National Institute on Aging/NIH

251 Bayview Blvd.

Baltimore, MD  21224-6825


Tel. 410-558-8173<tel:410-558-8173>

Fax  410-558-8236<tel:410-558-8236>

[hidden email]<mailto:[hidden email]><mailto:[hidden email]<mailto:[hidden email]>>


________________________________
From: Knecht, David <[hidden email]<mailto:[hidden email]>>
Sent: Wednesday, March 28, 2018 10:53:13 AM
To: [hidden email]<mailto:[hidden email]>
Subject: mounting media and anti-fade reagents

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Post images on https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFAg&c=JeTkUgVztGMmhKYjxsy2rfoWYibK1YmxXez1G3oNStg&r=RBx0-WJrAO5vwSOLNmFbqYvikvIZS5ns3-USwvMOuLo&m=_intRa89BmLCxHGeG_MoI8OfuqtSxU79ebDpHBN5Dgk&s=ngLTCD5oicUBC3RCSYKKi2eG5hQ9tvm0yD_v6bD7cWw&e= and include the link in your posting.
*****

I recently switched from fluorescein/rhodamine labels to Alexa 477/568.  Much to my surprise, the alexa dyes bleached very rapidly in a spinning disk microscope with just buffer as mounting media.  I then added my usual phenylene diamine/glycerol based mounting media and the fluorescence disappeared completely for both alexa dyes.  I found this problem well known in the literature.  So what do people used for home made mounting media cocktails for alexa dyes?  Alternatively, are there any dyes that work well with PDA?  I am tempted to just switch back to fluorescein and rhodamine.  Thanks- Dave

Dr. David Knecht
Professor , Department of Molecula<https://maps.google.com/?q=artment+of+Molecula&entry=gmail&source=g>r and Cell Biology University of Connecticut
91 N. Eagleville Rd.
U-3125
Storrs, CT 06269-3125
860-486-2200<tel:860-486-2200>

--
Alison J. North, Ph.D.,
Research Associate Professor and
Senior Director of the Bio-Imaging Resource <https://maps.google.com/?q=Director+of+the+Bio-Imaging+Resource+&entry=gmail&source=g> Center, The Rockefeller University,
1230 York Avenue,
New York,
NY 10065.
Tel: office     ++ 212 327 7488
Tel: lab        ++ 212 327 7486
Fax:            ++ 212 327 7489