Re: new prolong gold antifade with dapi **vendor reply**

[Kilgore, Jason A.]

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** Vendor Reply **

Hi, Pam,

It's actually not a new formulation.  We did more storage stability testing and found that ProLong Gold can be stored at RT without any effects on its performance, so we changed the storage temperature.  ProLong Diamond, on the other hands, is a new formulation (and should be stored at 4 degrees).  

Mounting in just PBS is a very good suggestion.

Another important test to troubleshoot your green background is to mount an empty slide with the PG mountant, with no sample whatsoever.  Is the green background still present?  If so, the product is defective and we should offer you a free replacement if it is within our 6-month guarantee period.  If the background isn't present, then it would suggest that the issue is related to the sample.

One cause of green background, independent of the mountant, may be that there wasn't sufficient washing of the sample after labeling with the FITC elastin, prior to mounting.

Another cause may be that your FITC conjugate may have an off-rate, where it is coming off of the specific label and leaching out into the background, due to low affinity.  This can be reduced somewhat by reducing the concentration of the conjugate, performing a post-fixation with formaldehyde (to crosslink the conjugate in place) prior to washing and mounting, storing the mounted slide at -20 (to slow the off-rate), or switching to a mountant that cures more firmly (such as Cytoseal, though that has drawbacks as well, such as the need for dehydrating the sample prior to mounting).

I hope this helps.  If you want more troubleshooting or need a replacement, please let me know offline.

Jason


Jason A. Kilgore
Technical Application Scientist
Molecular Probes Tech Support
Life Sciences Solutions

Thermo Fisher Scientific
29851 Willow Creek Rd.
Eugene, OR  97402-9132
1-800-955-6288 then option 4, then option 6, or
[phone]
[hidden email]
www.lifetechnologies.com

Please know that while Life Technologies is now a brand of Thermo Fisher Scientific, our ordering processes and representatives remain the same. We look forward to continuing to provide you with outstanding service and support.

This communication is intended solely for the individual/entity to whom it is addressed. It may contain confidential or legally privileged information.  Any unauthorized disclosure or copying is prohibited and may be unlawful. If you have received this communication in error, please notify the sender immediately and delete it from your system.


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Pamela Young
Sent: Thursday, July 17, 2014 1:00 AM
To: [hidden email]
Subject: new prolong gold antifade with dapi

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Hello Listers,

I have a user who just repeated an experiment for the millionth time but using the new formula of Prolong Gold Antifade with Dapi (P36935).  This new formula can be kept at room temp, but her former prolong had to be stored in the freezer.  As far as we can figure, the new prolong is the only thing changed in her protocol.  And unfortunately, instead of imaging beautiful FITC-labeled elastin, she saw green elastin and bright green cytosol.  She previously had seen in the FITC channel nothing from cells and green fibers (elastin) around the cells.

Has anyone tried the new prolong gold?  Because that is the only thing we can think of that might be different.  We are going to test by repeating and just mounting in PBS.

Thanks,
Pam

Dr Pamela A. Young | Light and Optical Microscopist Australian Centre for Microscopy & Microanalysis

THE UNIVERSITY OF SYDNEY
Rm 116A, Madsen Building F09 | The University of Sydney | NSW | 2006 | Australia T [phone] | F [phone] E [hidden email]<mailto:[hidden email]> | W http://sydney.edu.au/acmm

Incorporating:
Australian Microscopy & Microanalysis Research Facility (AMMRF) | W http://www.ammrf.org.au<http://www.ammrf.org.au/>
ARC Centre of Excellence for Design in Light Metals | W http://www.arclightmetals.org.au<http://www.arclightmetals.org.au/>

CRICOS 00026A
This email plus any attachments to it are confidential. Any unauthorised use is strictly prohibited. If you receive this email in error, please delete it and any attachments.

[Evelyn Ralston]

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Hi Jason,

I found this thread from last year and have more questions for you or others. We use Prolong Gold and let it cure, as recommended by Leica for STED microscopy. We are not sealing or refrigerating, so as to favor curing. But then, after the initial imaging, we put the slides in the fridge for later imaging, thinking that once PG is cured, that's irreversible, no? Well we find that after a few weeks in the fridge Prolong Gold is liquid again. If we keep the slides for 24h again at rm temp PG resolidifies.

Would this problem be avoided if we sealed with nail polish after curing or if we stored the slides at -20 instead of 4 degrees?


Evelyn Ralston, Ph.D.
Head, Light Imaging Section
Office of Science & Technology,NIAMS
National Institutes of Health,
Bldg 50, Rm 1535
Bethesda, MD 20892-8023
tel. [phone]




On Jul 17, 2014, at 12:45 PM, Kilgore, Jason 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.
> *****
>
> ** Vendor Reply **
>
> Hi, Pam,
>
> It's actually not a new formulation.  We did more storage stability testing and found that ProLong Gold can be stored at RT without any effects on its performance, so we changed the storage temperature.  ProLong Diamond, on the other hands, is a new formulation (and should be stored at 4 degrees).  
>
> Mounting in just PBS is a very good suggestion.
>
> Another important test to troubleshoot your green background is to mount an empty slide with the PG mountant, with no sample whatsoever.  Is the green background still present?  If so, the product is defective and we should offer you a free replacement if it is within our 6-month guarantee period.  If the background isn't present, then it would suggest that the issue is related to the sample.
>
> One cause of green background, independent of the mountant, may be that there wasn't sufficient washing of the sample after labeling with the FITC elastin, prior to mounting.
>
> Another cause may be that your FITC conjugate may have an off-rate, where it is coming off of the specific label and leaching out into the background, due to low affinity.  This can be reduced somewhat by reducing the concentration of the conjugate, performing a post-fixation with formaldehyde (to crosslink the conjugate in place) prior to washing and mounting, storing the mounted slide at -20 (to slow the off-rate), or switching to a mountant that cures more firmly (such as Cytoseal, though that has drawbacks as well, such as the need for dehydrating the sample prior to mounting).
>
> I hope this helps.  If you want more troubleshooting or need a replacement, please let me know offline.
>
> Jason
>
>
> Jason A. Kilgore
> Technical Application Scientist
> Molecular Probes Tech Support
> Life Sciences Solutions
>
> Thermo Fisher Scientific
> 29851 Willow Creek Rd.
> Eugene, OR  97402-9132
> 1-800-955-6288 then option 4, then option 6, or
> [phone]
> [hidden email]
> www.lifetechnologies.com
>
> Please know that while Life Technologies is now a brand of Thermo Fisher Scientific, our ordering processes and representatives remain the same. We look forward to continuing to provide you with outstanding service and support.
>
> This communication is intended solely for the individual/entity to whom it is addressed. It may contain confidential or legally privileged information.  Any unauthorized disclosure or copying is prohibited and may be unlawful. If you have received this communication in error, please notify the sender immediately and delete it from your system.
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Pamela Young
> Sent: Thursday, July 17, 2014 1:00 AM
> To: [hidden email]
> Subject: new prolong gold antifade with dapi
>
> *****
> 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.
> *****
>
> Hello Listers,
>
> I have a user who just repeated an experiment for the millionth time but using the new formula of Prolong Gold Antifade with Dapi (P36935).  This new formula can be kept at room temp, but her former prolong had to be stored in the freezer.  As far as we can figure, the new prolong is the only thing changed in her protocol.  And unfortunately, instead of imaging beautiful FITC-labeled elastin, she saw green elastin and bright green cytosol.  She previously had seen in the FITC channel nothing from cells and green fibers (elastin) around the cells.
>
> Has anyone tried the new prolong gold?  Because that is the only thing we can think of that might be different.  We are going to test by repeating and just mounting in PBS.
>
> Thanks,
> Pam
>
> Dr Pamela A. Young | Light and Optical Microscopist Australian Centre for Microscopy & Microanalysis
>
> THE UNIVERSITY OF SYDNEY
> Rm 116A, Madsen Building F09 | The University of Sydney | NSW | 2006 | Australia T [phone] | F [phone] E [hidden email]<mailto:[hidden email]> | W http://sydney.edu.au/acmm
>
> Incorporating:
> Australian Microscopy & Microanalysis Research Facility (AMMRF) | W http://www.ammrf.org.au<http://www.ammrf.org.au/>
> ARC Centre of Excellence for Design in Light Metals | W http://www.arclightmetals.org.au<http://www.arclightmetals.org.au/>
>
> CRICOS 00026A
> This email plus any attachments to it are confidential. Any unauthorised use is strictly prohibited. If you receive this email in error, please delete it and any attachments.

[Kilgore, Jason A.]

*****
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.
*****

Hi, Evelyn,

If the ProLong Gold is rehydrated it will become liquid again (for instance, you can dis-mount the coverslip by immersing in warm PBS for an hour or so).  My suspicion is that your refrigerator condition is very humid, leading to gradual rehydration.  4 degree temperature alone won't lead to this.  When you take it out to RT, it again dehydrates and cures.  If the freezer is humid, it could potentially happen there, too.

You could test this by taking some blank slides mounted with the ProLong Gold, with and without the sealing.

I think your idea of sealing it should help to prevent rehydration, as long as it is fully cured at the time of sealing.

You can seal with nail polish, but be aware that the solvents used can sometimes leach under the coverslip through the mountant and affect dyes - quenching GFP, for instance.  Instead, I would suggest a two-part hobby epoxy.

I hope this helps, but please let me know if you need anything further.

Jason



From: Evelyn Ralston [mailto:[hidden email]]
Sent: Thursday, June 18, 2015 10:43 AM
To: Confocal Microscopy List
Cc: [hidden email]
Subject: Re: prolong gold de- and re-solidifying

Hi Jason,

I found this thread from last year and have more questions for you or others. We use Prolong Gold and let it cure, as recommended by Leica for STED microscopy. We are not sealing or refrigerating, so as to favor curing. But then, after the initial imaging, we put the slides in the fridge for later imaging, thinking that once PG is cured, that's irreversible, no? Well we find that after a few weeks in the fridge Prolong Gold is liquid again. If we keep the slides for 24h again at rm temp PG resolidifies.

Would this problem be avoided if we sealed with nail polish after curing or if we stored the slides at -20 instead of 4 degrees?


Evelyn Ralston, Ph.D.
Head, Light Imaging Section
Office of Science & Technology,NIAMS
National Institutes of Health,
Bldg 50, Rm 1535
Bethesda, MD 20892-8023
tel. [phone]





On Jul 17, 2014, at 12:45 PM, Kilgore, Jason 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.
*****

** Vendor Reply **

Hi, Pam,

It's actually not a new formulation.  We did more storage stability testing and found that ProLong Gold can be stored at RT without any effects on its performance, so we changed the storage temperature.  ProLong Diamond, on the other hands, is a new formulation (and should be stored at 4 degrees).

Mounting in just PBS is a very good suggestion.

Another important test to troubleshoot your green background is to mount an empty slide with the PG mountant, with no sample whatsoever.  Is the green background still present?  If so, the product is defective and we should offer you a free replacement if it is within our 6-month guarantee period.  If the background isn't present, then it would suggest that the issue is related to the sample.

One cause of green background, independent of the mountant, may be that there wasn't sufficient washing of the sample after labeling with the FITC elastin, prior to mounting.

Another cause may be that your FITC conjugate may have an off-rate, where it is coming off of the specific label and leaching out into the background, due to low affinity.  This can be reduced somewhat by reducing the concentration of the conjugate, performing a post-fixation with formaldehyde (to crosslink the conjugate in place) prior to washing and mounting, storing the mounted slide at -20 (to slow the off-rate), or switching to a mountant that cures more firmly (such as Cytoseal, though that has drawbacks as well, such as the need for dehydrating the sample prior to mounting).

I hope this helps.  If you want more troubleshooting or need a replacement, please let me know offline.

Jason


Jason A. Kilgore
Technical Application Scientist
Molecular Probes Tech Support
Life Sciences Solutions

Thermo Fisher Scientific
29851 Willow Creek Rd.
Eugene, OR  97402-9132
1-800-955-6288 then option 4, then option 6, or
[phone]
[hidden email]<mailto:[hidden email]>
www.lifetechnologies.com<http://www.lifetechnologies.com>

Please know that while Life Technologies is now a brand of Thermo Fisher Scientific, our ordering processes and representatives remain the same. We look forward to continuing to provide you with outstanding service and support.

This communication is intended solely for the individual/entity to whom it is addressed. It may contain confidential or legally privileged information.  Any unauthorized disclosure or copying is prohibited and may be unlawful. If you have received this communication in error, please notify the sender immediately and delete it from your system.


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]<mailto:[mailto:[hidden email]]> On Behalf Of Pamela Young
Sent: Thursday, July 17, 2014 1:00 AM
To: [hidden email]<mailto:[hidden email]>
Subject: new prolong gold antifade with dapi

*****
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.
*****

Hello Listers,

I have a user who just repeated an experiment for the millionth time but using the new formula of Prolong Gold Antifade with Dapi (P36935).  This new formula can be kept at room temp, but her former prolong had to be stored in the freezer.  As far as we can figure, the new prolong is the only thing changed in her protocol.  And unfortunately, instead of imaging beautiful FITC-labeled elastin, she saw green elastin and bright green cytosol.  She previously had seen in the FITC channel nothing from cells and green fibers (elastin) around the cells.

Has anyone tried the new prolong gold?  Because that is the only thing we can think of that might be different.  We are going to test by repeating and just mounting in PBS.

Thanks,
Pam

Dr Pamela A. Young | Light and Optical Microscopist Australian Centre for Microscopy & Microanalysis

THE UNIVERSITY OF SYDNEY
Rm 116A, Madsen Building F09 | The University of Sydney | NSW | 2006 | Australia T [phone] | F [phone] E [hidden email]<mailto:[hidden email]<mailto:[hidden email]%3cmailto:[hidden email]>> | W http://sydney.edu.au/acmm

Incorporating:
Australian Microscopy & Microanalysis Research Facility (AMMRF) | W http://www.ammrf.org.au<http://www.ammrf.org.au/<http://www.ammrf.org.au%3chttp:/www.ammrf.org.au/>>
ARC Centre of Excellence for Design in Light Metals | W http://www.arclightmetals.org.au<http://www.arclightmetals.org.au/<http://www.arclightmetals.org.au%3chttp:/www.arclightmetals.org.au/>>

CRICOS 00026A
This email plus any attachments to it are confidential. Any unauthorised use is strictly prohibited. If you receive this email in error, please delete it and any attachments.