Membrane Marker

[Sue Penrhyn-Lowe]

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Does anyone know of a surface membrane marker that does not internalise
during fixation or even a surface membrane marker  that is not internalised
when staining the membrane of fixed cells?
Thanks Sue

[mmodel]

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Lectins can be labeled in some cells. -

Mike Model
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From: Confocal Microscopy List [[hidden email]] on behalf of Sue Penrhyn-Lowe [[hidden email]]
Sent: Friday, May 31, 2013 5:45 AM
To: [hidden email]
Subject: Membrane Marker

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Does anyone know of a surface membrane marker that does not internalise
during fixation or even a surface membrane marker  that is not internalised
when staining the membrane of fixed cells?
Thanks Sue

[mmodel]

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I meant, fluorescent lectins can be used to label the surface
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Sue Penrhyn-Lowe [[hidden email]]
Sent: Friday, May 31, 2013 5:45 AM
To: [hidden email]
Subject: Membrane Marker

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Does anyone know of a surface membrane marker that does not internalise
during fixation or even a surface membrane marker  that is not internalised
when staining the membrane of fixed cells?
Thanks Sue

[Kelly Lundsten-2]

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Yes, my thought was lectin or simply using NHS ester reactive dye directly.  The internalization you are seeing I would suspect is from using DiI-like probes, where even if they do contain chloromethyl groups, the cross linking to the cell surface with fixation is pretty inefficient.  Also, using too much DiI-type probe can lead to internalization and hence labeling of internal membrane-bound compartments... and it's hard to control the amount of probe used.

Live/Dead Fixable viability kits are already provided at the right molar ratio of dye to label the surface of the cell.  But, they do need to be live when labeled, but fixing them will not cause any internalization of the probe.

Just a thought.

Kelly


On May 31, 2013, at 12:52 PM, "MODEL, MICHAEL" <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I meant, fluorescent lectins can be used to label the surface
> ________________________________________
> From: Confocal Microscopy List [[hidden email]] on behalf of Sue Penrhyn-Lowe [[hidden email]]
> Sent: Friday, May 31, 2013 5:45 AM
> To: [hidden email]
> Subject: Membrane Marker
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Does anyone know of a surface membrane marker that does not internalise
> during fixation or even a surface membrane marker  that is not internalised
> when staining the membrane of fixed cells?
> Thanks Sue
>

[Tim Feinstein-2]

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The Na/K ATPase makes a nice and specific plasma membrane marker, but you need to find a decent antibody for it.  There is one for dog that works well in MDCK cells.  

cheers,


TF

Timothy Feinstein, PhD
Visiting Research Associate
Laboratory for GPCR Biology
Dept. of Pharmacology & Chemical Biology
University of Pittsburgh, School of Medicine
BST W1301, 200 Lothrop St.
Pittsburgh, PA  15261

On May 31, 2013, at 6:59 AM, Kelly Lundsten wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Yes, my thought was lectin or simply using NHS ester reactive dye directly.  The internalization you are seeing I would suspect is from using DiI-like probes, where even if they do contain chloromethyl groups, the cross linking to the cell surface with fixation is pretty inefficient.  Also, using too much DiI-type probe can lead to internalization and hence labeling of internal membrane-bound compartments... and it's hard to control the amount of probe used.
>
> Live/Dead Fixable viability kits are already provided at the right molar ratio of dye to label the surface of the cell.  But, they do need to be live when labeled, but fixing them will not cause any internalization of the probe.
>
> Just a thought.
>
> Kelly
>
>
> On May 31, 2013, at 12:52 PM, "MODEL, MICHAEL" <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> I meant, fluorescent lectins can be used to label the surface
>> ________________________________________
>> From: Confocal Microscopy List [[hidden email]] on behalf of Sue Penrhyn-Lowe [[hidden email]]
>> Sent: Friday, May 31, 2013 5:45 AM
>> To: [hidden email]
>> Subject: Membrane Marker
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Does anyone know of a surface membrane marker that does not internalise
>> during fixation or even a surface membrane marker  that is not internalised
>> when staining the membrane of fixed cells?
>> Thanks Sue
>>

[Martin Wessendorf-2]

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Dear Sue--

On 5/31/2013 4:45 AM, Sue Penrhyn-Lowe wrote:

> Does anyone know of a surface membrane marker that does not internalise
> during fixation or even a surface membrane marker  that is not internalised
> when staining the membrane of fixed cells?

In addition to lectin markers, the NaK-ATPase may be an option--it's in
the plasma membrane, if not solely in the membrane.  You can get an
antibody against it for minimal cost from the Developmental Studies
Hybridoma Bank: http://dshb.biology.uiowa.edu/ .  We used the alpha5
antibody (http://dshb.biology.uiowa.edu/a5) in rat spinal cord and it
worked well (http://www.ncbi.nlm.nih.gov/pubmed/20974701).

Good luck!

Martin Wessendorf

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Kilgore, Jason-2]

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** Vendor reply **

There are two options I'd recommend:  CellMask plasma membrane stains (of which there are orange or far red options) or wheat germ agglutinin dye conjugates (of which a full range of dye options are available).  Both can be used to label live and then fix, or can label already formaldehyde-fixed cells.  

CellMask PM dyes are more uniform, but will not survive subsequent permeabilization, whereas WGA conjugates will survive (but you must label BEFORE permeabilization or you will also label internal structures).

Jason

Molecular Probes Tech Support



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sue Penrhyn-Lowe
Sent: Friday, May 31, 2013 2:45 AM
To: [hidden email]
Subject: Membrane Marker

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Does anyone know of a surface membrane marker that does not internalise
during fixation or even a surface membrane marker  that is not internalised
when staining the membrane of fixed cells?
Thanks Sue