detection of secreted cytokines by immunofluorescence

[Julio Vazquez]

Dear List, 

One of our customers would like to detect secreted (extracellular) cytokines in tissue sections by immunofluorescence microscopy against a protein tag on the secreted molecule. Could any of you provide a protocol or other information (suitable tag, etc..) on how to achieve this? I can't find much info in the literature, and don't even know if this is feasible. many thanks, 

Julio.

--

Julio Vazquez

Fred Hutchinson Cancer Research Center

1100 Fairview Ave. N.,  mailstop DE-512

Seattle, WA 98109-1024

http://www.fhcrc.org/ 

[Paul Rigby-2]

Hi All,
Depending upon exactly what questions are being asked, I would look at the Luminex and related technologies. A simple Google search will bring up many links. This is not imaging but is a quantitative technique (based on flow cytometry) that can work very well for secreted (soluble) cytokines, particularly if several different cytokines or peptides need to be assayed in the same sample.
Cheers
Paul
 
Dr Paul Rigby
Associate Professor
Centre for Microscopy, Characterisation & Analysis (M510)
The University of Western Australia
35 Stirling Highway
Crawley WA 6009
Phone [phone]

________________________________

From: Confocal Microscopy List on behalf of Julio Vazquez
Sent: Fri 4/30/2010 12:29 PM
To: [hidden email]
Subject: detection of secreted cytokines by immunofluorescence


Dear List,  

One of our customers would like to detect secreted (extracellular) cytokines in tissue sections by immunofluorescence microscopy against a protein tag on the secreted molecule. Could any of you provide a protocol or other information (suitable tag, etc..) on how to achieve this? I can't find much info in the literature, and don't even know if this is feasible. many thanks,

Julio.


--
Julio Vazquez
Fred Hutchinson Cancer Research Center
1100 Fairview Ave. N.,  mailstop DE-512
Seattle, WA 98109-1024

http://www.fhcrc.org <http://www.fhcrc.org/> /

[Carol Heckman]

julio-
The usual way is cryosectioning or vibratome, but in this case, it sounds as though the protein would diffuse away from the section before one was able to view it.  The only realistic way may be to embed in a bio-compatible polymer and view the sections by wide-field fluorescence microscopy.  I don't think i have ever seen a procedure for doing this.
carol heckman
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Julio Vazquez [[hidden email]]
Sent: Friday, April 30, 2010 12:29 PM
To: [hidden email]
Subject: detection of secreted cytokines by immunofluorescence

Dear List,

One of our customers would like to detect secreted (extracellular) cytokines in tissue sections by immunofluorescence microscopy against a protein tag on the secreted molecule. Could any of you provide a protocol or other information (suitable tag, etc..) on how to achieve this? I can't find much info in the literature, and don't even know if this is feasible. many thanks,

Julio.

--
Julio Vazquez
Fred Hutchinson Cancer Research Center
1100 Fairview Ave. N.,  mailstop DE-512
Seattle, WA 98109-1024

http://www.fhcrc.org/

[George McNamara]

Hi Julio,

Consider adapting ELIspot / FluoroSPOT ... or even simply starting out with this.

Handbook of ELISPOT - Methods and Protocols
Series: Methods in Molecular Biology, Vol. 302
Kalyuzhny, Alexander E. (Ed.)
2005, 336 p. 123 illus., 10 in color., Hardcover
ISBN: 978-1-58829-469-2
online PDFs at http://www.springerlink.com/content/978-1-58829-469-2

FluoroSPOT (not called as such by Cyntellect) is the front end for the LEAP CellXpress application, http://www.cyntellect.com/content/products/leap/cellxpress.cfm

I have been trying to get Zeiss and Leica to add these capabilities to ZEN and LAS AF (or at least Matrix M3), respectively, since I have confocal microscopes from each with powerful enough lasers that we could "confocal LEAP" a multiwell plate every night. Obviously the confocal software would need enough image analysis capability to make the measurements (currently lacking in ZEN and LAS AF!), then optionally laser ablate the unwanted cells. Anyone who thinks this is a good idea - please lobby your industry contacts.

Sincerely,

George



At 12:29 PM 4/30/2010, Julio Vazquez wrote:

Dear List,

One of our customers would like to detect secreted (extracellular) cytokines in tissue sections by immunofluorescence microscopy against a protein tag on the secreted molecule. Could any of you provide a protocol or other information (suitable tag, etc..) on how to achieve this? I can't find much info in the literature, and don't even know if this is feasible. many thanks,

Julio.

--
Julio Vazquez
Fred Hutchinson Cancer Research Center
1100 Fairview Ave. N.,  mailstop DE-512
Seattle, WA 98109-1024

http://www.fhcrc.org/

George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine
Miami, FL 33136
[hidden email]
[hidden email]
305-243-8436 office
http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+ spectra .xlsx file is in the zip file)
http://home.earthlink.net/~geomcnamara