generic membrane marker for FFPE tissue?

[Cromey, Douglas W - (dcromey)]

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I am working with a lab that has been trying to use Cadhedrin as a generic membrane marker for formalin-fixed, paraffin sectioned rat liver.  They are not having much luck.  Is there a good fixed tissue membrane marker that works reliably on paraffin sectioned tissue that you can recommend?

Because of ongoing problems with autofluorescence, it would be highly preferable if the membrane fluorescence was not in the typical FITC/GFP emission range, we would highly prefer something more red-shifted.

Doug

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Douglas W. Cromey, M.S. - Associate Scientific Investigator
Dept. of Cellular & Molecular Medicine, University of Arizona
1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

office:  AHSC 4212         email: [hidden email]
voice:  520-626-2824       fax:  [phone]

http://swehsc.pharmacy.arizona.edu/micro
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[David Crossman]

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I would try Wheat germ agglutinin  (WGA). It binds to glycosylated proteins found in the cell membrane and extra cellular matrix. It is commonly used for as cell surface marker. Its advantage is that tends not to be sensitive to fixation as antibody targets can be. You can purchase it conjugated with a range of fluorophores (eg 488, 568, 647 etc). Simply dilute the stock in PBS and incubate for couple of hours (try 1 in 50 dilution as a starting point). I typically include it with the secondary antibodies in my labelling runs.

Cheers,
David


David Crossman PhD
Biophysics & Biophotonics Research Group
Department of Physiology
Faculty of Medical and Health Sciences
The University of Auckland
Private Bag 92019
Auckland 1142
New Zealand
Phone [phone] ext 89964
Fax: [phone] attn: D Crossman
Email: [hidden email]


________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Cromey, Douglas W - (dcromey) [[hidden email]]
Sent: Thursday, August 15, 2013 8:58 AM
To: [hidden email]
Subject: generic membrane marker for FFPE tissue?

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I am working with a lab that has been trying to use Cadhedrin as a generic membrane marker for formalin-fixed, paraffin sectioned rat liver.  They are not having much luck.  Is there a good fixed tissue membrane marker that works reliably on paraffin sectioned tissue that you can recommend?

Because of ongoing problems with autofluorescence, it would be highly preferable if the membrane fluorescence was not in the typical FITC/GFP emission range, we would highly prefer something more red-shifted.

Doug

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Douglas W. Cromey, M.S. - Associate Scientific Investigator
Dept. of Cellular & Molecular Medicine, University of Arizona
1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

office:  AHSC 4212         email: [hidden email]
voice:  520-626-2824       fax:  [phone]

http://swehsc.pharmacy.arizona.edu/micro
Home of: "Microscopy and Imaging Resources on the WWW"

[Kilgore, Jason-2]

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** Vendor Reply **

I haven't had any luck with WGA in paraffin sections.  But even in cryosections, it isn't selective for PM in my hands.  Works great for PM in live cells or in formaldehyde-fixed cells which have not yet been permeabilized.

I don't know of any other organic dyes or protein conjugates that would work in this situation.

The only solution I could suggest is to identify a different antigen to target with an antibody.

Cheers,

Jason


Jason A. Kilgore
Technical Application Scientist
Molecular Probes Labeling and Detection Technologies
Cells Systems Division
 
T 1 800 955 6288 then option 4, then option 6,  or  541 335 0353 * F 541 335 0238
29851 Willow Creek Rd * Eugene * OR * 97402-9132 * United States

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Crossman
Sent: Wednesday, August 14, 2013 2:37 PM
To: [hidden email]
Subject: Re: generic membrane marker for FFPE tissue?

*****
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I would try Wheat germ agglutinin  (WGA). It binds to glycosylated proteins found in the cell membrane and extra cellular matrix. It is commonly used for as cell surface marker. Its advantage is that tends not to be sensitive to fixation as antibody targets can be. You can purchase it conjugated with a range of fluorophores (eg 488, 568, 647 etc). Simply dilute the stock in PBS and incubate for couple of hours (try 1 in 50 dilution as a starting point). I typically include it with the secondary antibodies in my labelling runs.

Cheers,
David


David Crossman PhD
Biophysics & Biophotonics Research Group
Department of Physiology
Faculty of Medical and Health Sciences
The University of Auckland
Private Bag 92019
Auckland 1142
New Zealand
Phone [phone] ext 89964
Fax: [phone] attn: D Crossman
Email: [hidden email]


________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Cromey, Douglas W - (dcromey) [[hidden email]]
Sent: Thursday, August 15, 2013 8:58 AM
To: [hidden email]
Subject: generic membrane marker for FFPE tissue?

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To join, leave or search the confocal microscopy listserv, go to:
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I am working with a lab that has been trying to use Cadhedrin as a generic membrane marker for formalin-fixed, paraffin sectioned rat liver.  They are not having much luck.  Is there a good fixed tissue membrane marker that works reliably on paraffin sectioned tissue that you can recommend?

Because of ongoing problems with autofluorescence, it would be highly preferable if the membrane fluorescence was not in the typical FITC/GFP emission range, we would highly prefer something more red-shifted.

Doug

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Douglas W. Cromey, M.S. - Associate Scientific Investigator
Dept. of Cellular & Molecular Medicine, University of Arizona
1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

office:  AHSC 4212         email: [hidden email]
voice:  520-626-2824       fax:  [phone]

http://swehsc.pharmacy.arizona.edu/micro
Home of: "Microscopy and Imaging Resources on the WWW"

[George McNamara]

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Hi Doug,

Beta-catenin should work well for plasma membrane. Can also be detected
in other parts of cells if certain signaling pathways were active.

Human Protein Atlas has data, and several (polyclonal) antisera. Most
IHC vendors should have (monoclonal) antibodies to it.
http://www.proteinatlas.org/ENSG00000168036

*CTNNB1 <http://www.proteinatlas.org/ENSG00000168036>

*see, for example this tissue microarray core of a normal pancreas (can
zoom up about 2x online)
http://www.proteinatlas.org/images/29159/62947_A_2_3.jpg

immunofluorescence of cells in culture are at
http://www.proteinatlas.org/ENSG00000168036/subcellular

Full credit to Prof. Vesa Kaartinen, who showed me awesome beta-catenin
plasma membrane labeling of a tissue section (sorry, don't remember what
body part, or species - probably mouse, maybe palate), when we were at
Childrens Hospital Los Angeles. Vesa is now at U. Michigan School of
Dentistry.

Enjoy,

George



On 8/14/2013 3:58 PM, Cromey, Douglas W - (dcromey) wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I am working with a lab that has been trying to use Cadhedrin as a generic membrane marker for formalin-fixed, paraffin sectioned rat liver.  They are not having much luck.  Is there a good fixed tissue membrane marker that works reliably on paraffin sectioned tissue that you can recommend?
>
> Because of ongoing problems with autofluorescence, it would be highly preferable if the membrane fluorescence was not in the typical FITC/GFP emission range, we would highly prefer something more red-shifted.
>
> Doug
>
> ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
> Douglas W. Cromey, M.S. - Associate Scientific Investigator
> Dept. of Cellular&  Molecular Medicine, University of Arizona
> 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>
> office:  AHSC 4212         email:[hidden email]
> voice:  520-626-2824       fax:  [phone]
>
> http://swehsc.pharmacy.arizona.edu/micro
> Home of: "Microscopy and Imaging Resources on the WWW"
>
>    

--



George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
http://works.bepress.com/gmcnamara/26/