mcherry and fading

[Boswell, Carl A - (cboswell)]

Dear listers,

We have fruit fly imaginal discs labelled with mCherry, and find that live
material maintains robust fluorescence when imaging with a Zeiss 510
confocal, but fixed material (standard 3% PFA) fades rapidly.  I'm struck by
this disparity in that my experience with mCherry was that it was stable
(compared with DsRed) regardless of the condition.

Any thoughts?

Thanks,

carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

[Gregg Sobocinski]

Carl,
My immediate suspect is your mounting medium. There are a few basic compositions used for mounting medium, and most fluorophores have a preference. I don't have any further details, but a call to a vendor could get  you the information you need. Hopefully, someone with mCherry experience can chime in.

Good luck. I've been in that situation.
~Gregg

Gregg Sobocinski
Microscope Imaging Specialist
University of Michigan, MCDB Dept.
Ann Arbor, Michigan
USA



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Carl Boswell
Sent: Friday, February 05, 2010 12:55 PM
To: [hidden email]
Subject: mcherry and fading

Dear listers,

We have fruit fly imaginal discs labelled with mCherry, and find that live
material maintains robust fluorescence when imaging with a Zeiss 510
confocal, but fixed material (standard 3% PFA) fades rapidly.  I'm struck by
this disparity in that my experience with mCherry was that it was stable
(compared with DsRed) regardless of the condition.

Any thoughts?

Thanks,

carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

[Vitaly Boyko]

Yes, I have seen it. Instead, I used 0.5% PFA for 20' for fixation of virus like particles.

However, during winter, as humidity in the lab is usually "unhealthyly" very low, fading could be also caused by the sample over drying up upon mounting. I used ProlongGold for mounting, it was usually OK during spring and fall months.

 

Vitaly

 

---

From: "Sobocinski, Gregg" <[hidden email]>
To: [hidden email]
Sent: Fri, February 5, 2010 3:51:53 PM
Subject: Re: mCherry and fading

Carl,
My immediate suspect is your mounting medium. There are a few basic compositions used for mounting medium, and most fluorophores have a preference. I don't have any further details, but a call to a vendor could get  you the information you need. Hopefully, someone with mCherry experience can chime in.

Good luck. I've been in that situation.
~Gregg

Gregg Sobocinski
Microscope Imaging Specialist
University of Michigan, MCDB Dept.
Ann Arbor, Michigan
USA



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Carl Boswell
Sent: Friday, February 05, 2010 12:55 PM
To: [hidden email]
Subject: mcherry and fading

Dear listers,

We have fruit fly imaginal discs labelled with mCherry, and find that live
material maintains robust fluorescence when imaging with a Zeiss 510
confocal, but fixed material (standard 3% PFA) fades rapidly.  I'm struck by
this disparity in that my experience with mCherry was that it was stable
(compared with DsRed) regardless of the condition.

Any thoughts?

Thanks,

carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]