Fluorescent Dye and Labeling Fibronectin

[Jonathan Stricker]

Hello,
I am looking for suggestions for a good bright fluorescent dye that is  
not in the usual red or green excitation/emission wavelengths,  
something either between red and green or a little shorter wavelength  
than green. I am attempting to visualize fibronectin; what methods  
have people had success visualizing fibronectin? I have had some  
success labeling the fibronectin, but the dye I used (Alexa 405) is  
not very bright, and I was wondering if anyone has ever had luck  
labeling fibronectin, or labeling an antibody to fibronectin? Any  
advice on these two questions would be much appreciated!

Jonathan Stricker
University of Chicago

[Melinda Larsen]

I have successfully used the MP kit for labeling human plasma
fibronectin with both Alexa 647 and Alexa 488. The Alexa 568 kit was
not as successful for unknown reasons and I didn't try any other
probes. This was published in Larsen, et al. 2006. JCS. 19:3376-3384
using methods originally used in Pankov, et al. 2000. JCB.
148:1075-1090. I didn't label FN antibodies, but the same kit can
theoretically be used to do this.

Melinda Larsen
University at Albany, SUNY

On Wed, Dec 3, 2008 at 11:06 AM, Jonathan Stricker
<[hidden email]> wrote:

> Hello,
> I am looking for suggestions for a good bright fluorescent dye that is not
> in the usual red or green excitation/emission wavelengths, something either
> between red and green or a little shorter wavelength than green. I am
> attempting to visualize fibronectin; what methods have people had success
> visualizing fibronectin? I have had some success labeling the fibronectin,
> but the dye I used (Alexa 405) is not very bright, and I was wondering if
> anyone has ever had luck labeling fibronectin, or labeling an antibody to
> fibronectin? Any advice on these two questions would be much appreciated!
>
> Jonathan Stricker
> University of Chicago
>

[Gens, John Scott]

Jonathan-

Could you supply a little more detail about your experiment?  What
organism are you working with? Are you thinking about looking at fixed
tissue or following  localization in live cells?

This probably isn't important as far as color selection is concerned
but it can make a big difference in which fibronectin antibody you'd
want to conjugate it to, whether bleaching is a concern, or whether you
can negate bleaching by adding an anti-fade reagent.


Scott

--
J. Scott Gens, Ph.D.
Biocomplexity Institute
Indiana University Physics
Room 024, Swain West Bldg.
Bloomington IN 47405
812-855-7946
[hidden email]


Quoting Jonathan Stricker <[hidden email]>:

> Hello,
> I am looking for suggestions for a good bright fluorescent dye that
> is  not in the usual red or green excitation/emission wavelengths,
> something either between red and green or a little shorter wavelength
>  than green. I am attempting to visualize fibronectin; what methods
> have people had success visualizing fibronectin? I have had some
> success labeling the fibronectin, but the dye I used (Alexa 405) is
> not very bright, and I was wondering if anyone has ever had luck
> labeling fibronectin, or labeling an antibody to fibronectin? Any
> advice on these two questions would be much appreciated!
>
> Jonathan Stricker
> University of Chicago
>

[Ignatius, Mike]

Hi Jonathan,

You mentioned that AF 405 was not very bright.  AF 405 is not as
photostable as other dyes in that range.  While a tad brighter, it
bleaches too fast.  Fine for Flow, but not for imaging. We have three
dyes, AF405, Marina Blue and Pacific Blue, all excited by 405 light.
For imaging, especially when using an antifade mountant, Marina Blue is
the clear winner, showing twice the signal strength after 80 seconds of
continuous imaging.  You may want to try that.

If you or anyone else wants an excel spread sheet of these values send
me a note off line.  Too bad the list-serve isn't file share-friendly or
I could merely show the data.  

We also have an AF 430 dye if you have the right light source.

Mike Ignatius,

Molecular Probes, now a part of Life Technologies

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Jonathan Stricker
Sent: Wednesday, December 03, 2008 8:06 AM
To: [hidden email]
Subject: Fluorescent Dye and Labeling Fibronectin

Hello,
I am looking for suggestions for a good bright fluorescent dye that is  
not in the usual red or green excitation/emission wavelengths,  
something either between red and green or a little shorter wavelength  
than green. I am attempting to visualize fibronectin; what methods  
have people had success visualizing fibronectin? I have had some  
success labeling the fibronectin, but the dye I used (Alexa 405) is  
not very bright, and I was wondering if anyone has ever had luck  
labeling fibronectin, or labeling an antibody to fibronectin? Any  
advice on these two questions would be much appreciated!

Jonathan Stricker
University of Chicago

[Sam's Mail]

How would you describe the photo characteristics of AF 430 with 440 nm excitation as compared to Marina Blue with 405nm excitation?

I would be interested if the data would indicate one of them vs. the other for live cell aqueous or fixed Prolong Gold style work.

I would appreciate the Excel sheet as well Mike.

Thanks!
--
Samuel A. Connell
Director of Light Microscopy
Cell & Tissue Imaging Center
St. Jude Children's Research Hospital
262 Danny Thomas Place
Memphis, TN 38105-3678
Office [phone]
Cell [phone]
[hidden email]
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Ignatius, Mike [[hidden email]]
Sent: Wednesday, December 03, 2008 3:53 PM
To: [hidden email]
Subject: Re: Fluorescent Dye and Labeling Fibronectin ***Vendor Response***.        .

Hi Jonathan,

You mentioned that AF 405 was not very bright.  AF 405 is not as
photostable as other dyes in that range.  While a tad brighter, it
bleaches too fast.  Fine for Flow, but not for imaging. We have three
dyes, AF405, Marina Blue and Pacific Blue, all excited by 405 light.
For imaging, especially when using an antifade mountant, Marina Blue is
the clear winner, showing twice the signal strength after 80 seconds of
continuous imaging.  You may want to try that.

If you or anyone else wants an excel spread sheet of these values send
me a note off line.  Too bad the list-serve isn't file share-friendly or
I could merely show the data.

We also have an AF 430 dye if you have the right light source.

Mike Ignatius,

Molecular Probes, now a part of Life Technologies

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Jonathan Stricker
Sent: Wednesday, December 03, 2008 8:06 AM
To: [hidden email]
Subject: Fluorescent Dye and Labeling Fibronectin

Hello,
I am looking for suggestions for a good bright fluorescent dye that is
not in the usual red or green excitation/emission wavelengths,
something either between red and green or a little shorter wavelength
than green. I am attempting to visualize fibronectin; what methods
have people had success visualizing fibronectin? I have had some
success labeling the fibronectin, but the dye I used (Alexa 405) is
not very bright, and I was wondering if anyone has ever had luck
labeling fibronectin, or labeling an antibody to fibronectin? Any
advice on these two questions would be much appreciated!

Jonathan Stricker
University of Chicago

[Ignatius, Mike]

Hello Dr. Connell,

AlexaFluor 430 is certainly the best for 440nm ex. Pac Blue has a
slightly broad red shifted shoulder, but really not much at 440nm, less
than 10%.  Our web site has a spectraviewer where you can toggle on/off
ex/ems for up to five dyes at once.  Including the analysis you
requested.  

http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluores
cence-SpectraViewer.html

The excel sheet which I will send under separate cover shows the PBS vs
PROLONG GOLD properties.  Pac Blue starts out brighter, but eventually
with continued imaging in either PBS or Prolong Gold, Marina wins out
over Alexa.  (Fun aside: nomenclature derives from Alex and Marina, who
are the son and daughter of Probes founders, Drs. Richard and Rosario
Haugland.)

Kind Regards,

Mike Ignatius

Molecular Probes, Now a part of Life Technologies.  

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Connell, Samuel
Sent: Wednesday, December 03, 2008 8:11 PM
To: [hidden email]
Subject: Re: Fluorescent Dye and Labeling Fibronectin ***Vendor
Response***. .

How would you describe the photo characteristics of AF 430 with 440 nm
excitation as compared to Marina Blue with 405nm excitation?

I would be interested if the data would indicate one of them vs. the
other for live cell aqueous or fixed Prolong Gold style work.

I would appreciate the Excel sheet as well Mike.

Thanks!
--
Samuel A. Connell
Director of Light Microscopy
Cell & Tissue Imaging Center
St. Jude Children's Research Hospital
262 Danny Thomas Place
Memphis, TN 38105-3678
Office [phone]
Cell [phone]
[hidden email]
________________________________________
From: Confocal Microscopy List [[hidden email]] On
Behalf Of Ignatius, Mike [[hidden email]]
Sent: Wednesday, December 03, 2008 3:53 PM
To: [hidden email]
Subject: Re: Fluorescent Dye and Labeling Fibronectin ***Vendor
Response***.        .

Hi Jonathan,

You mentioned that AF 405 was not very bright.  AF 405 is not as
photostable as other dyes in that range.  While a tad brighter, it
bleaches too fast.  Fine for Flow, but not for imaging. We have three
dyes, AF405, Marina Blue and Pacific Blue, all excited by 405 light.
For imaging, especially when using an antifade mountant, Marina Blue is
the clear winner, showing twice the signal strength after 80 seconds of
continuous imaging.  You may want to try that.

If you or anyone else wants an excel spread sheet of these values send
me a note off line.  Too bad the list-serve isn't file share-friendly or
I could merely show the data.

We also have an AF 430 dye if you have the right light source.

Mike Ignatius,

Molecular Probes, now a part of Life Technologies

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Jonathan Stricker
Sent: Wednesday, December 03, 2008 8:06 AM
To: [hidden email]
Subject: Fluorescent Dye and Labeling Fibronectin

Hello,
I am looking for suggestions for a good bright fluorescent dye that is
not in the usual red or green excitation/emission wavelengths,
something either between red and green or a little shorter wavelength
than green. I am attempting to visualize fibronectin; what methods
have people had success visualizing fibronectin? I have had some
success labeling the fibronectin, but the dye I used (Alexa 405) is
not very bright, and I was wondering if anyone has ever had luck
labeling fibronectin, or labeling an antibody to fibronectin? Any
advice on these two questions would be much appreciated!

Jonathan Stricker
University of Chicago

[Adrian Smith-6]

Following up an old post...

(trying to set up the best four colour combination for SP5 imaging -  
will send a separate email about that overall question).

Looking at the Invitrogen spectralviewer it appears that Marina Blue  
has only small % excitation at 405 while Pacific Blue is almost 100%?

I assume that contributes to the difference in initial brightness but  
the stability of Marina Blue wins out in the end for prolonged imaging?

In any case, Mike, if you are still reading, I would also like a copy  
of the spreadsheet.

Regards,

Adrian

On 05/12/2008, at 6:25 AM, Ignatius, Mike wrote:

> Hello Dr. Connell,
>
> AlexaFluor 430 is certainly the best for 440nm ex. Pac Blue has a
> slightly broad red shifted shoulder, but really not much at 440nm,  
> less
> than 10%.  Our web site has a spectraviewer where you can toggle on/
> off
> ex/ems for up to five dyes at once.  Including the analysis you
> requested.
>
> http://www.invitrogen.com/site/us/en/home/support/Research-Tools/Fluores
> cence-SpectraViewer.html
>
> The excel sheet which I will send under separate cover shows the PBS  
> vs
> PROLONG GOLD properties.  Pac Blue starts out brighter, but eventually
> with continued imaging in either PBS or Prolong Gold, Marina wins out
> over Alexa.  (Fun aside: nomenclature derives from Alex and Marina,  
> who
> are the son and daughter of Probes founders, Drs. Richard and Rosario
> Haugland.)
>
> Kind Regards,
>
> Mike Ignatius
>
> Molecular Probes, Now a part of Life Technologies.
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]
> ]
> On Behalf Of Connell, Samuel
> Sent: Wednesday, December 03, 2008 8:11 PM
> To: [hidden email]
> Subject: Re: Fluorescent Dye and Labeling Fibronectin ***Vendor
> Response***. .
>
> How would you describe the photo characteristics of AF 430 with 440 nm
> excitation as compared to Marina Blue with 405nm excitation?
>
> I would be interested if the data would indicate one of them vs. the
> other for live cell aqueous or fixed Prolong Gold style work.
>
> I would appreciate the Excel sheet as well Mike.
>
> Thanks!
> --
> Samuel A. Connell
> Director of Light Microscopy
> Cell & Tissue Imaging Center
> St. Jude Children's Research Hospital
> 262 Danny Thomas Place
> Memphis, TN 38105-3678
> Office [phone]
> Cell [phone]
> [hidden email]
> ________________________________________
> From: Confocal Microscopy List [[hidden email]] On
> Behalf Of Ignatius, Mike [[hidden email]]
> Sent: Wednesday, December 03, 2008 3:53 PM
> To: [hidden email]
> Subject: Re: Fluorescent Dye and Labeling Fibronectin ***Vendor
> Response***.        .
>
> Hi Jonathan,
>
> You mentioned that AF 405 was not very bright.  AF 405 is not as
> photostable as other dyes in that range.  While a tad brighter, it
> bleaches too fast.  Fine for Flow, but not for imaging. We have three
> dyes, AF405, Marina Blue and Pacific Blue, all excited by 405 light.
> For imaging, especially when using an antifade mountant, Marina Blue  
> is
> the clear winner, showing twice the signal strength after 80 seconds  
> of
> continuous imaging.  You may want to try that.
>
> If you or anyone else wants an excel spread sheet of these values send
> me a note off line.  Too bad the list-serve isn't file share-
> friendly or
> I could merely show the data.
>
> We also have an AF 430 dye if you have the right light source.
>
> Mike Ignatius,
>
> Molecular Probes, now a part of Life Technologies
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]
> ]
> On Behalf Of Jonathan Stricker
> Sent: Wednesday, December 03, 2008 8:06 AM
> To: [hidden email]
> Subject: Fluorescent Dye and Labeling Fibronectin
>
> Hello,
> I am looking for suggestions for a good bright fluorescent dye that is
> not in the usual red or green excitation/emission wavelengths,
> something either between red and green or a little shorter wavelength
> than green. I am attempting to visualize fibronectin; what methods
> have people had success visualizing fibronectin? I have had some
> success labeling the fibronectin, but the dye I used (Alexa 405) is
> not very bright, and I was wondering if anyone has ever had luck
> labeling fibronectin, or labeling an antibody to fibronectin? Any
> advice on these two questions would be much appreciated!
>
> Jonathan Stricker
> University of Chicago

[kirk-20]

Dear Listserv,

Our University is exploring the possibility of purchasing a Laboratory
Information Management System (LIMS) for our centralized core
facilities. There appear to be many commercial solutions out there, but
at least in a quick review many are targeted toward industry. We would
use this LIMS for core facilities including proteomics,
genomics/sequencing, imaging, flow cytometry, etc. Thus, there will be
need in flexibility for each core. We need functionality from simple
scheduling to work flow, data storage and sharing, search,
accounting/billing and a simple to use and web-based interface for
investigators to access.

Does anyone know if such an integrated system exists and as important is
it compatible with an academic environment?

Thanks in advance for any suggestions. Vendors are welcome to contact me
directly.

Best Regards, Kirk

Kirk J. Czymmek, Ph.D.
Associate Professor
Department of Biological Sciences
University of Delaware
Delaware Biotechnology Institute Bio-Imaging Center, Director
Newark, DE 19711
[phone]