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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:[log in to unmask]] On Behalf Of David Crossman
Sent: Wednesday, August 14, 2013 2:37 PM
To: [log in to unmask]
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 (64)-9-3737599 ext 89964
Fax: (64)-9-373 7499 attn: D Crossman
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From: Confocal Microscopy List [[log in to unmask]] on behalf of Cromey, Douglas W - (dcromey) [[log in to unmask]]
Sent: Thursday, August 15, 2013 8:58 AM
To: [log in to unmask]
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
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Douglas W. Cromey, M.S. - Associate Scientific Investigator
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