Re: mounting media and anti-fade reagents **vendor reply**

[Kilgore, Jason A.]

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** vendor reply **

When I was in R&D here at Molecular Probes, I helped develop antifades here at Molecular Probes, including assisting with ProLong and SlowFade Gold and advising on the "Diamond" versions.  As part of that, we evaluated the homebrew antifade components. DABCO did pretty poorly (except for fluorescein), and, as Alison mentioned, can quench some dyes. P-phenylene diamine (PPD) is very poor, too, and has blue autofluorescence (as does Vectashield products, which I suspect has some version of PPD in it, which we have also noted kills AF647 and other similar dyes. and can seriously quench AF594).

As others have pointed out, ProLong Diamond and SlowFade Diamond improved upon the "Gold" versions mainly for fluorescent proteins and BODIPY dyes.  We now also have ProLong Glass, which has an RI like that of glass and thus has better resolution. I highly recommend these for Alexa Fluor dyes.

If you don't want to cure the mountant (such as for 3D imaging), I recommend using the SlowFade version (SlowFade Diamond) since it doesn't cure and already has a high RI, though you will need to seal the edges (I recommend melted paraffin painted on with a cotton-tipped applicator).

Jason

Jason A. Kilgore
Technical Application Scientist
Molecular Probes / EVOS Tech Support
Thermo Fisher Scientific
 
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Alison North
Sent: Wednesday, March 28, 2018 8:43 AM
To: [hidden email]
Subject: Re: mounting media and anti-fade reagents

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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
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>
> Biomedical Research Center (BRC) Rm 8B135
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> National Institute on Aging/NIH
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> 251 Bayview Blvd.
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> Baltimore, MD  21224-6825
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> ________________________________
> From: Knecht, David <[hidden email]>
> Sent: Wednesday, March 28, 2018 10:53:13 AM
> To: [hidden email]
> Subject: 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

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