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Suggestions for 4 colour stain (on Leica SP5)

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mmodel

Re: Whole Cell Dye

A paper has been published on this subject: "Comparison of reagents for shape analysis of fixed cells by automated fluorescence microscopy", Cytometry 52A, 90-100, 2003

-----Original Message-----
From: Cameron Nowell [mailto:[hidden email]]
Sent: August 6, 2009 7:38 PM
To: [hidden email]
Subject: Whole Cell Dye

Hi List

Can anyone recommend a dye (or antibody) that will stain the whole cell
evenly to the plasma membrane? I need a dye that will give fairly even
staining across the whole cell to make segmentation easier.

I have tried CellMask but is it a bit variable in its staining pattern,
there are blobs in the cytoplasm and nucleus may or may not stain up.
Something that stains just the plasma membrane would also be suitible. I
have used the invitrogen FX dyes before (not for this experiemnt) and
have found them a bit hit and miss too. I have also tried WGA but it
doesn't give an even stain around the mebrane which makes segmentation
quite dificult.

Any suggestions would be greatly appreciated.

Thanks

Cam

Cameron J. Nowell
Microscopy Manager
Central Resource for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
Office: +61 3 9341 3155
Mobile: +61422882700
Fax: +61 3 9341 3104
Facility Website

This communication is intended only for the named recipient and may contain
information that is confidential, legally privileged or subject to
copyright; the Ludwig Institute for Cancer Research Ltd does not waiver any
rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not
necessarily reflect the views of the Ludwig Institute for Cancer Research
Ltd.

Cameron Nowell

Re: Whole Cell Dye

Thanks Michael. Nice paper but unfortunately it only deals with single
cells that are nice and flat. We are trying to segment confluent layers
of cells that are fairly tall.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of MODEL, MICHAEL
Sent: Wednesday, 12 August 2009 12:21 AM
To: [hidden email]
Subject: Re: Whole Cell Dye

A paper has been published on this subject: "Comparison of reagents for
shape analysis of fixed cells by automated fluorescence microscopy",
Cytometry 52A, 90-100, 2003

-----Original Message-----
From: Cameron Nowell [mailto:[hidden email]]
Sent: August 6, 2009 7:38 PM
To: [hidden email]
Subject: Whole Cell Dye

Hi List

Can anyone recommend a dye (or antibody) that will stain the whole cell
evenly to the plasma membrane? I need a dye that will give fairly even
staining across the whole cell to make segmentation easier.

I have tried CellMask but is it a bit variable in its staining pattern,
there are blobs in the cytoplasm and nucleus may or may not stain up.
Something that stains just the plasma membrane would also be suitible. I
have used the invitrogen FX dyes before (not for this experiemnt) and
have found them a bit hit and miss too. I have also tried WGA but it
doesn't give an even stain around the mebrane which makes segmentation
quite dificult.

Any suggestions would be greatly appreciated.

Thanks

Cam

Cameron J. Nowell
Microscopy Manager
Central Resource for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
Office: +61 3 9341 3155
Mobile: +61422882700
Fax: +61 3 9341 3104
Facility Website

This communication is intended only for the named recipient and may
contain
information that is confidential, legally privileged or subject to
copyright; the Ludwig Institute for Cancer Research Ltd does not waiver
any
rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do
not
necessarily reflect the views of the Ludwig Institute for Cancer
Research
Ltd.

No virus found in this incoming message.
Checked by AVG - www.avg.com
Version: 8.5.392 / Virus Database: 270.13.49/2295 - Release Date:
08/10/09 18:19:00

This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research Ltd does not waiver any rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research Ltd.

axel k preuss

Re: Whole Cell Dye

How about the live cell stains like SYTO (Mol Probes). Membrane stain would be R 18?
-axel

--- On Tue, 8/11/09, Cameron Nowell <[hidden email]> wrote:

> From: Cameron Nowell <[hidden email]>
> Subject: Re: Whole Cell Dye
> To: [hidden email]
> Date: Tuesday, August 11, 2009, 11:33 PM
> Thanks Michael. Nice paper but
> unfortunately it only deals with single
> cells that are nice and flat. We are trying to segment
> confluent layers
> of cells that are fairly tall.
>
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of MODEL, MICHAEL
> Sent: Wednesday, 12 August 2009 12:21 AM
> To: [hidden email]
> Subject: Re: Whole Cell Dye
>
> A paper has been published on this subject: "Comparison of
> reagents for
> shape analysis of fixed cells by automated fluorescence
> microscopy",
> Cytometry 52A, 90-100, 2003
>
>
>
> -----Original Message-----
> From: Cameron Nowell [mailto:[hidden email]]
> Sent: August 6, 2009 7:38 PM
> To: [hidden email]
> Subject: Whole Cell Dye
>
> Hi List
>
> Can anyone recommend a dye (or antibody) that will stain
> the whole cell
> evenly to the plasma membrane? I need a dye that will give
> fairly even
> staining across the whole cell to make segmentation
> easier.
>
> I have tried CellMask but is it a bit variable in its
> staining pattern,
> there are blobs in the cytoplasm and nucleus may or may not
> stain up.
> Something that stains just the plasma membrane would also
> be suitible. I
> have used the invitrogen FX dyes before (not for this
> experiemnt) and
> have found them a bit hit and miss too. I have also tried
> WGA but it
> doesn't give an even stain around the mebrane which makes
> segmentation
> quite dificult.
>
> Any suggestions would be greatly appreciated.
>
>
> Thanks
>
> Cam
>
>
> Cameron J. Nowell
> Microscopy Manager
> Central Resource for Advanced Microscopy
> Ludwig Institute for Cancer Research
> PO Box 2008
> Royal Melbourne Hospital
> Victoria, 3050
> AUSTRALIA
> Office: +61 3 9341 3155
> Mobile: +61422882700
> Fax: +61 3 9341 3104
> Facility Website
>
>
>
>
> This communication is intended only for the named recipient
> and may
> contain
> information that is confidential, legally privileged or
> subject to
> copyright; the Ludwig Institute for Cancer Research Ltd
> does not waiver
> any
> rights if you have received this communication in error.
> The views expressed in this communication are those of the
> sender and do
> not
> necessarily reflect the views of the Ludwig Institute for
> Cancer
> Research
> Ltd.
>
> No virus found in this incoming message.
> Checked by AVG - www.avg.com
> Version: 8.5.392 / Virus Database: 270.13.49/2295 - Release
> Date:
> 08/10/09 18:19:00
>
>
> This communication is intended only for the named recipient
> and may contain information that is confidential, legally
> privileged or subject to copyright; the Ludwig Institute for
> Cancer Research Ltd does not waiver any rights if you have
> received this communication in error.
> The views expressed in this communication are those of the
> sender and do not necessarily reflect the views of the
> Ludwig Institute for Cancer Research Ltd.
>
>

Arne K Christensen

Re: Whole Cell Dye. .

In reply to this post by Sam's Mail

The Developmental Studies Hybridoma Bank has a nice monoclonal antibody
against the alpha subunit of the NKAtpase. It's called alpha5 (a5).

http://dshb.biology.uiowa.edu/a5

Connell, Samuel wrote:

> Tim,
>
> Do you have a particular Ab to recommend for the sodium-potassium ATPase you mentioned?
>
> 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 (901) 595-2536
> Cell (901) 603-3162
> [hidden email]
> ________________________________________
> From: Confocal Microscopy List [[hidden email]] On Behalf Of Timothy Feinstein [[hidden email]]
> Sent: Thursday, August 06, 2009 8:41 PM
> To: [hidden email]
> Subject: Re: Whole Cell Dye. .
>
> Hi Cam,
>
> In the past we have had good results with chloromethylfluorescein
> diacetate (CMFDA). CMFDA is an intravital dye that fluoresces in the same
> channel as GFP, however as far as I know only works in live cells. If
> transfection is possible, people often use GFP-family proteins as a
> co-transfection marker. These pass freely through nuclear pores and
> therefore produce even cellular labeling. If an antibody is preferable,
> the sodium-potassium ATPase marks the plasma membrane as clearly and
> evenly as anything that I have seen.
>
> All the best,
>
>
> Tim
>
> Timothy Feinstein, PhD
> Postdoctoral Associate, Vilardaga laboratory
> University of Pittsburgh Dept. of Pharmacology and Chemical Biology
> Pittsburgh, PA
>
>
>
>> Hi List
>>
>> Can anyone recommend a dye (or antibody) that will stain the whole cell
>> evenly to the plasma membrane? I need a dye that will give fairly even
>> staining across the whole cell to make segmentation easier.
>>
>> I have tried CellMask but is it a bit variable in its staining pattern,
>> there are blobs in the cytoplasm and nucleus may or may not stain up.
>> Something that stains just the plasma membrane would also be suitible. I
>> have used the invitrogen FX dyes before (not for this experiemnt) and
>> have found them a bit hit and miss too. I have also tried WGA but it
>> doesn't give an even stain around the mebrane which makes segmentation
>> quite dificult.
>>
>> Any suggestions would be greatly appreciated.
>>
>>
>> Thanks
>>
>> Cam
>>
>>
>> Cameron J. Nowell
>> Microscopy Manager
>> Central Resource for Advanced Microscopy
>> Ludwig Institute for Cancer Research
>> PO Box 2008
>> Royal Melbourne Hospital
>> Victoria, 3050
>> AUSTRALIA
>> Office: +61 3 9341 3155
>> Mobile: +61422882700
>> Fax: +61 3 9341 3104
>> Facility Website
>>
>>
>>
>>
>> This communication is intended only for the named recipient and may
>> contain information that is confidential, legally privileged or subject to
>> copyright; the Ludwig Institute for Cancer Research Ltd does not waiver
>> any rights if you have received this communication in error.
>> The views expressed in this communication are those of the sender and do
>> not necessarily reflect the views of the Ludwig Institute for Cancer
>> Research Ltd.
>>
>>
>>
>
>
> Email Disclaimer: www.stjude.org/emaildisclaimer
>
>

--

__

Arne K Christensen

Postdoctoral Research Associate
University of Massachusetts, Amherst

USGS Conte Anadromous Fish Research Center
One Migratory Way, PO Box 796
Turners Falls, MA 01376

Email: [hidden email]
Phone: (413) 863-3827
Fax: (413) 863-9810
URL: www.biobog.com

Sam's Mail

Re: Whole Cell Dye. .. .

Thanks for the suggestion Arne. Have you used this Ab for marking plasma membranes for segmentation before? What has been your experience with this reagent?

Much appreciate the feedback.

Best,

--
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 (901) 595-2536
Cell (901) 603-3162
[hidden email]

On 8/17/09 10:43 AM, "Arne K Christensen" <[hidden email]> wrote:

The Developmental Studies Hybridoma Bank has a nice monoclonal antibody
against the alpha subunit of the NKAtpase. It's called alpha5 (a5).

http://dshb.biology.uiowa.edu/a5

Connell, Samuel wrote: