p-Phenylenediamine anti-fade reagent (modified protocol)

[obsydion]

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Hi all,

Just wondering if anyone has had much experience with using
p-Phenylenediamine (PPD) as an anti-fade reagent? Rather than incorporate it
into a mounting medium, I wish to use it in a different way:

I have a fixed tissue slice/section that I wish to image WITHOUT a coverslip
using a water immersion lens. Would it be possible to make up some PPD and
add it to the PBS that I will then immerse the sample in for imaging? Most
PPD protocols I came across instruct that the it be made up in glycerol but
I assume that is purely so you can use it directly as a mounting medium.

So to reiterate: Can I make up PPD in PBS (no glycerol) and then use it as
my immersion solution to image a NON-coverslipped tissue slice/section?

Cheers,
Kelvin

[mcammer]

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I don't know carcinogenic it really is, but we used to treat it very very carefully, like powdered DAB.

_________________________________________
Michael Cammer, Assistant Research Scientist
Skirball Institute of Biomolecular Medicine
Lab: [phone]  Cell: [phone]

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Kelvin Poon [[hidden email]]
Sent: Monday, March 11, 2013 6:56 PM
To: [hidden email]
Subject: p-Phenylenediamine anti-fade reagent (modified protocol)

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Hi all,

Just wondering if anyone has had much experience with using
p-Phenylenediamine (PPD) as an anti-fade reagent? Rather than incorporate it
into a mounting medium, I wish to use it in a different way:

I have a fixed tissue slice/section that I wish to image WITHOUT a coverslip
using a water immersion lens. Would it be possible to make up some PPD and
add it to the PBS that I will then immerse the sample in for imaging? Most
PPD protocols I came across instruct that the it be made up in glycerol but
I assume that is purely so you can use it directly as a mounting medium.

So to reiterate: Can I make up PPD in PBS (no glycerol) and then use it as
my immersion solution to image a NON-coverslipped tissue slice/section?

Cheers,
Kelvin

[Knecht, David]

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We do this routinely for cells in glass bottom dishes fixed in situ.  We make up the PPD in buffer shortly before use.  The only issue is that the PPD will oxidize with time and the solution will turn orange.  We have not had that affect imaging in relatively short term situations (several days in fridge).  It is not as high resolution since the refractive index does not match glass or oil, but that depends on how critical your imaging needs are.  Dave

On Mar 11, 2013, at 6:56 PM, Kelvin Poon wrote:

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Hi all,

Just wondering if anyone has had much experience with using
p-Phenylenediamine (PPD) as an anti-fade reagent? Rather than incorporate it
into a mounting medium, I wish to use it in a different way:

I have a fixed tissue slice/section that I wish to image WITHOUT a coverslip
using a water immersion lens. Would it be possible to make up some PPD and
add it to the PBS that I will then immerse the sample in for imaging? Most
PPD protocols I came across instruct that the it be made up in glycerol but
I assume that is purely so you can use it directly as a mounting medium.

So to reiterate: Can I make up PPD in PBS (no glycerol) and then use it as
my immersion solution to image a NON-coverslipped tissue slice/section?

Cheers,
Kelvin

David Knecht, Ph.D.
Professor and Head of Microscopy Facility
Department of Molecular and Cell Biology
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)

[Tim Feinstein-2]

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Hi David,

You could use trolox, a nontoxic but less potent antioxidant/antifade that we use in hydrous solutions (~300 uM) when fading is a major issue.  

Keep in mind that PPD interferes with cy dyes (cy3, cy5 etc), so avoid using those together.  

Cheers,


TF

Timothy Feinstein, Ph.D.
Visiting Research Associate
Laboratory for GPCR Biology
Dept. of Pharmacology & Chemical Biology
University of Pittsburgh, School of Medicine
BST W1301, 200 Lothrop St.
Pittsburgh, PA  15261

On Mar 11, 2013, at 9:01 PM, "Knecht, David" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> We do this routinely for cells in glass bottom dishes fixed in situ.  We make up the PPD in buffer shortly before use.  The only issue is that the PPD will oxidize with time and the solution will turn orange.  We have not had that affect imaging in relatively short term situations (several days in fridge).  It is not as high resolution since the refractive index does not match glass or oil, but that depends on how critical your imaging needs are.  Dave
>
> On Mar 11, 2013, at 6:56 PM, Kelvin Poon wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi all,
>
> Just wondering if anyone has had much experience with using
> p-Phenylenediamine (PPD) as an anti-fade reagent? Rather than incorporate it
> into a mounting medium, I wish to use it in a different way:
>
> I have a fixed tissue slice/section that I wish to image WITHOUT a coverslip
> using a water immersion lens. Would it be possible to make up some PPD and
> add it to the PBS that I will then immerse the sample in for imaging? Most
> PPD protocols I came across instruct that the it be made up in glycerol but
> I assume that is purely so you can use it directly as a mounting medium.
>
> So to reiterate: Can I make up PPD in PBS (no glycerol) and then use it as
> my immersion solution to image a NON-coverslipped tissue slice/section?
>
> Cheers,
> Kelvin
>
> David Knecht, Ph.D.
> Professor and Head of Microscopy Facility
> Department of Molecular and Cell Biology
> U-3125
> 91 N. Eagleville Rd.
> University of Connecticut
> Storrs, CT 06269
> 860-486-2200
> 860-486-4331 (fax)