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Suggestions for 4 colour stain (on Leica SP5)

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Adrian Smith-6

Suggestions for 4 colour stain (on Leica SP5)

Hi all,

We are trying to set up a four colour combination for tissues sections
on our SP5 and are looking for recommendations.

The only colour that is currently "fixed" is AlexaFluor 488 (there is
a difficult reagent that is working well and the researchers don't
want to change unless they absolutely have to).

We have 405, multi-line Argon, 561 and 633 lasers on a Leica SP5.

We are considering the following and would appreciate comments:-

* Pacific Blue or Marina Blue (probably Marina based on Mike Ignatius'
comments on and off this list)

* AlexaFluor 488

* AlexaFluor 546 or 555 or 594
- not sure which of these will be best from the point of view of (i)
brightness, (ii) photostability and (iii) overlap with the other dyes
(eg from the spectral viewer it looks like the 546 will be slightly
excited by the 488 laser for AF488, while the red dyes (below) will be
slightly excited with 561)
- interestingly Molecular Probes include 555 in the some of their
triple label kits - I'm wondering why they have chosen that as 546
looks at least comparable on the spectral viewer (obviously more going
on - as I hope Mike will reveal ;)

* AlexaFluor 647 or 700
- my experience with AF700 with 633nm excitation in flow has not be
fantastic but it can be a useful dye there and it certainly has less
overlap with 546 or 594 - no idea how it performs for confocal.

OR
Qdot 705 or Qdot 800
- I believe the SP5 will detect out to 800 so a lot of Qdot800 will
be lost but there is still a good part available according to the
spectral viewer.

Currently the researchers are leaning towards Marina Blue, AF488,
AF546, AF647.... anyone have a better idea?

Regards,

Adrian

Rosemary.White

Re: Suggestions for 4 colour stain (on Leica SP5)

You can separate Cy5 and Cy5.5 on the SP2, EX 633, EM 640-690 for Cy5, EM
690-800 for Cy5.5, so should also be fine on SP5. I know these numbers are
"wrong" for these dyes, but they work.
cheers,
Rosemary

On 6/08/09 2:57 PM, "Adrian Smith" <[hidden email]> wrote:

> Hi all,
>
> We are trying to set up a four colour combination for tissues sections
> on our SP5 and are looking for recommendations.
>
> The only colour that is currently "fixed" is AlexaFluor 488 (there is
> a difficult reagent that is working well and the researchers don't
> want to change unless they absolutely have to).
>
> We have 405, multi-line Argon, 561 and 633 lasers on a Leica SP5.
>
> We are considering the following and would appreciate comments:-
>
> * Pacific Blue or Marina Blue (probably Marina based on Mike Ignatius'
> comments on and off this list)
>
> * AlexaFluor 488
>
> * AlexaFluor 546 or 555 or 594
> - not sure which of these will be best from the point of view of (i)
> brightness, (ii) photostability and (iii) overlap with the other dyes
> (eg from the spectral viewer it looks like the 546 will be slightly
> excited by the 488 laser for AF488, while the red dyes (below) will be
> slightly excited with 561)
> - interestingly Molecular Probes include 555 in the some of their
> triple label kits - I'm wondering why they have chosen that as 546
> looks at least comparable on the spectral viewer (obviously more going
> on - as I hope Mike will reveal ;)
>
> * AlexaFluor 647 or 700
> - my experience with AF700 with 633nm excitation in flow has not be
> fantastic but it can be a useful dye there and it certainly has less
> overlap with 546 or 594 - no idea how it performs for confocal.
>
> OR
> Qdot 705 or Qdot 800
> - I believe the SP5 will detect out to 800 so a lot of Qdot800 will
> be lost but there is still a good part available according to the
> spectral viewer.
>
>
> Currently the researchers are leaning towards Marina Blue, AF488,
> AF546, AF647.... anyone have a better idea?
>
> Regards,
>
> Adrian

Cameron, Lisa

Re: Suggestions for 4 colour stain (on Leica SP5)

In reply to this post by Adrian Smith-6

If you have a 561 laser, I'd recommend Alexa568 in place of Alexa546, 555 or
594.

- Lisa

-----Original Message-----
From: Confocal Microscopy List on behalf of Adrian Smith
Sent: Thu 8/6/2009 12:57 AM
To: [hidden email]
Subject: [CONFOCALMICROSCOPY] Suggestions for 4 colour stain (on Leica SP5)

Hi all,

We are trying to set up a four colour combination for tissues sections
on our SP5 and are looking for recommendations.

The only colour that is currently "fixed" is AlexaFluor 488 (there is
a difficult reagent that is working well and the researchers don't
want to change unless they absolutely have to).

We have 405, multi-line Argon, 561 and 633 lasers on a Leica SP5.

We are considering the following and would appreciate comments:-

* Pacific Blue or Marina Blue (probably Marina based on Mike Ignatius'
comments on and off this list)

* AlexaFluor 488

* AlexaFluor 546 or 555 or 594
- not sure which of these will be best from the point of view of (i)
brightness, (ii) photostability and (iii) overlap with the other dyes
(eg from the spectral viewer it looks like the 546 will be slightly
excited by the 488 laser for AF488, while the red dyes (below) will be
slightly excited with 561)
- interestingly Molecular Probes include 555 in the some of their
triple label kits - I'm wondering why they have chosen that as 546
looks at least comparable on the spectral viewer (obviously more going
on - as I hope Mike will reveal ;)

* AlexaFluor 647 or 700
- my experience with AF700 with 633nm excitation in flow has not be
fantastic but it can be a useful dye there and it certainly has less
overlap with 546 or 594 - no idea how it performs for confocal.

OR
Qdot 705 or Qdot 800
- I believe the SP5 will detect out to 800 so a lot of Qdot800 will
be lost but there is still a good part available according to the
spectral viewer.

Currently the researchers are leaning towards Marina Blue, AF488,
AF546, AF647.... anyone have a better idea?

Regards,

Adrian

The information in this e-mail is intended only for the person to whom it is
addressed. If you believe this e-mail was sent to you in error and the e-mail
contains patient information, please contact the Partners Compliance HelpLine at
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dispose of the e-mail.

lechristophe

Re: Suggestions for 4 colour stain (on Leica SP5)

Adrian,

Have a look at the DyLight405 secondary antibodies for 405nm excitation. They are a bit difficult to find (Jackson should sell them at some point, for now Rockland has them in a wide variety of species). In my hands they work reasonnably well as "blue" dyes, and have more species diversity than Alexa405 sold by Invitrogen/Molecular Probes. I didn't test Pacific or Marina Blue, for the same reason (species variety). When you get to four different antibodies, that means usually four different species (for example mouse/rabbit/rat/chicken). Usually the you put "general markers" (organelle, structural, cytoskeleton...) as the blue channel rather than your faint, hard to find antibody for your protein of study that you put in the green or red channel to maximize image quality. That antibody usually is mouse or rabbit, so you end up using "general markers" from more exotic species (chicken, goat, rat...) and this is the point where you need a wide variety of 4055 secondary antibodies (anti-exotic species + not made in goat in case you have a goat primary).

Christophe

On Thu, Aug 6, 2009 at 7:57 AM, Cameron, Lisa <[hidden email]> wrote:

If you have a 561 laser, I'd recommend Alexa568 in place of Alexa546, 555 or
594.

- Lisa

-----Original Message-----
From: Confocal Microscopy List on behalf of Adrian Smith
Sent: Thu 8/6/2009 12:57 AM
To: [hidden email]
Subject: [CONFOCALMICROSCOPY] Suggestions for 4 colour stain (on Leica SP5)

Hi all,

We are trying to set up a four colour combination for tissues sections
on our SP5 and are looking for recommendations.

The only colour that is currently "fixed" is AlexaFluor 488 (there is
a difficult reagent that is working well and the researchers don't
want to change unless they absolutely have to).

We have 405, multi-line Argon, 561 and 633 lasers on a Leica SP5.

We are considering the following and would appreciate comments:-

* Pacific Blue or Marina Blue (probably Marina based on Mike Ignatius'
comments on and off this list)

* AlexaFluor 488

* AlexaFluor 546 or 555 or 594
- not sure which of these will be best from the point of view of (i)
brightness, (ii) photostability and (iii) overlap with the other dyes
(eg from the spectral viewer it looks like the 546 will be slightly
excited by the 488 laser for AF488, while the red dyes (below) will be
slightly excited with 561)
- interestingly Molecular Probes include 555 in the some of their
triple label kits - I'm wondering why they have chosen that as 546
looks at least comparable on the spectral viewer (obviously more going
on - as I hope Mike will reveal ;)

* AlexaFluor 647 or 700
- my experience with AF700 with 633nm excitation in flow has not be
fantastic but it can be a useful dye there and it certainly has less
overlap with 546 or 594 - no idea how it performs for confocal.

OR
Qdot 705 or Qdot 800
- I believe the SP5 will detect out to 800 so a lot of Qdot800 will
be lost but there is still a good part available according to the
spectral viewer.

Currently the researchers are leaning towards Marina Blue, AF488,
AF546, AF647.... anyone have a better idea?

Regards,

Adrian

The information in this e-mail is intended only for the person to whom it is
addressed. If you believe this e-mail was sent to you in error and the e-mail
contains patient information, please contact the Partners Compliance HelpLine at
http://www.partners.org/complianceline . If the e-mail was sent to you in error
but does not contain patient information, please contact the sender and properly
dispose of the e-mail.

Marc Thibault

Re: Suggestions for 4 colour stain (on Leica SP5)

In reply to this post by Adrian Smith-6

Here is a ref. for an article from our group that did 4 color imaging in 2006.
In it, you will find other references pertaining to that. Hope this helps.

Migration of bone marrow stromal cells in 3D: 4 color methodology reveals
spatially and temporally coordinated events.

Thibault MM, Buschmann MD.

Cell Motil Cytoskeleton. 2006 Dec;63(12):725-40

Selon Adrian Smith <[hidden email]>:

Glen MacDonald-2

Re: Suggestions for 4 colour stain (on Leica SP5)

In reply to this post by Adrian Smith-6

Dear Adrian

the AF546 is included to accommodate the 543 nm HeNe laser.

As suggested already, AF568 is your best 'red' option. The AF594
seems a bit brighter, but it will bleed into the far red channel, and,
if using sequential capture, you will find that it excites with the
633 nm line sufficiently to appear in the far red channel.

Regards,
Glen

Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923 USA
(206) 616-4156
[hidden email]

******************************************************************************
The box said "Requires WindowsXP or better", so I bought a Macintosh.
******************************************************************************

On Aug 5, 2009, at 9:57 PM, Adrian Smith wrote:

>
> * AlexaFluor 546 or 555 or 594
> - not sure which of these will be best from the point of view of
> (i) brightness, (ii) photostability and (iii) overlap with the other
> dyes (eg from the spectral viewer it looks like the 546 will be
> slightly excited by the 488 laser for AF488, while the red dyes
> (below) will be slightly excited with 561)
> - interestingly Molecular Probes include 555 in the some of their
> triple label kits - I'm wondering why they have chosen that as 546
> looks at least comparable on the spectral viewer (obviously more
> going on - as I hope Mike will reveal ;)

B. Prabhakar Pandian

Transwell chambers

In reply to this post by lechristophe

Hello everybody,
Can anyone point to vendors who might be able to
give some samples of the transwell chambers. Also if there is any good
paper that compares all the transwell chambers, that will be great.
We want to see which chambers will be good to coat with extracellular
matrix with different gelatin concentrations and then do TEER measurements.

Thanks,

-Prabhakar

Cameron Nowell

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.

Timothy Feinstein

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.
>
>

JOEL B. SHEFFIELD

Re: Whole Cell Dye

In reply to this post by Cameron Nowell

Have you tried eosin? It seems to be a general stain for most cultured cells, and it fluoresces in the red.
Joel

On Thu, Aug 6, 2009 at 7:37 PM, Cameron Nowell <[hidden email]> wrote:

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.

--

Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL: http://astro.temple.edu/~jbs

Sam's Mail

Re: Whole Cell Dye. .

In reply to this post by Timothy Feinstein

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

Email Disclaimer: www.stjude.org/emaildisclaimer

Tim O'Brien Sr.

Re: Whole Cell Dye

In reply to this post by Cameron Nowell

I just tried the Vybrant DiD fluorescent cell stain (Invirtogen) for
the same purpose in adherent cultured cells, and I found the labeling
to be patchy. Tech support said to try adding it in serum-free
medium as the dye bound to proteins in serum and the proteins were
unevenly taken up. I tried that and the stain was still pretty
punctate. I will try some other cell types, and report back if I can
achieve an even plasma membrane stain.

Tim O'Brien
CISMM, UNC Physics and AStronomy

On Aug 6, 2009, at 7:37 PM, Cameron Nowell wrote:

> 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.
>

Aryeh Weiss

Re: Whole Cell Dye

In reply to this post by Cameron Nowell

I do this with live cells by labeling the medium. Try using an anionic
dye like sulforhodamine B (SRB) in the medium . It will not enter
healthy cells. In a confocal scope, the inside of the cells will be
dark. About 1 uM in solution works very well. You can get a very nice
segmentation. SRB is really cheap -- aobut $80 for 5g. 5g is enough to
last many lifetimes...

--aryeh

Cameron Nowell wrote:

--
Aryeh Weiss
School of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph: 972-3-5317638
FAX: 972-3-7384050

Adrian Smith-6

Re: Suggestions for 4 colour stain (on Leica SP5)

In reply to this post by Glen MacDonald-2

re: AF546 versus AF555 versus AF568

Sorry - I should have included AF568 in my list of options along with
AF555 and AF546.

However, according to the spectral viewer both AF546 and AF555 will be
excited more efficiently by the 561nm than AF568 (note the the peak
excitation for AF546 is actually shown around 560nm, whereas AF568
peak is shown closer to 580nm).

What the spectral viewer doesn't show of course is the relative
brightness and photostability... which I'm hoping someone has looked
at and can comment on.

Regards,

Adrian

On 07/08/2009, at 4:16 AM, Glen MacDonald wrote:

> Dear Adrian
>
> the AF546 is included to accommodate the 543 nm HeNe laser.
>
> As suggested already, AF568 is your best 'red' option. The AF594
> seems a bit brighter, but it will bleed into the far red channel,
> and, if using sequential capture, you will find that it excites with
> the 633 nm line sufficiently to appear in the far red channel.
>
>
> Regards,
> Glen
>
> Glen MacDonald
> Core for Communication Research
> Virginia Merrill Bloedel Hearing Research Center
> Box 357923
> University of Washington
> Seattle, WA 98195-7923 USA
> (206) 616-4156
> [hidden email]
>
> ******************************************************************************
> The box said "Requires WindowsXP or better", so I bought a Macintosh.
> ******************************************************************************
>
>
> On Aug 5, 2009, at 9:57 PM, Adrian Smith wrote:
>
>>
>> * AlexaFluor 546 or 555 or 594
>> - not sure which of these will be best from the point of view of
>> (i) brightness, (ii) photostability and (iii) overlap with the
>> other dyes (eg from the spectral viewer it looks like the 546 will
>> be slightly excited by the 488 laser for AF488, while the red dyes
>> (below) will be slightly excited with 561)
>> - interestingly Molecular Probes include 555 in the some of their
>> triple label kits - I'm wondering why they have chosen that as 546
>> looks at least comparable on the spectral viewer (obviously more
>> going on - as I hope Mike will reveal ;)

Jacqueline Ross

Re: Suggestions for 4 colour stain (on Leica SP5)

Hi Adrian,

In my experience, Alexa 568 is excited very well by the 561nm DPSS as is
the Alexa 594. I would choose Alexa 568 over 555 or 546 due to less
bleedthrough into the green (Alexa 488 channel). This is also the reason
that I sometimes recommend Alexa 594 if people are imaging using a
fluorescence microscope with conventional longpass barrier filters.

Kind regards,

Jacqui

Jacqueline Ross

Biomedical Imaging Microscopist
Biomedical Imaging Research Unit
School of Medical Sciences
Faculty of Medical & Health Sciences
The University of Auckland
Private Bag 92019
Auckland, NEW ZEALAND

Tel: 64 9 373 7599 Ext 87438
Fax: 64 9 373 7484

http://www.fmhs.auckland.ac.nz/sms/biru/

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Adrian Smith
Sent: Friday, 7 August 2009 4:51 p.m.
To: [hidden email]
Subject: Re: Suggestions for 4 colour stain (on Leica SP5)

re: AF546 versus AF555 versus AF568

Sorry - I should have included AF568 in my list of options along with
AF555 and AF546.

However, according to the spectral viewer both AF546 and AF555 will be
excited more efficiently by the 561nm than AF568 (note the the peak
excitation for AF546 is actually shown around 560nm, whereas AF568
peak is shown closer to 580nm).

What the spectral viewer doesn't show of course is the relative
brightness and photostability... which I'm hoping someone has looked
at and can comment on.

Regards,

Adrian

On 07/08/2009, at 4:16 AM, Glen MacDonald wrote:

************************************************************************
******
> The box said "Requires WindowsXP or better", so I bought a Macintosh.
>
************************************************************************
******
>
>
> On Aug 5, 2009, at 9:57 PM, Adrian Smith wrote:
>
>>
>> * AlexaFluor 546 or 555 or 594
>> - not sure which of these will be best from the point of view of

>> (i) brightness, (ii) photostability and (iii) overlap with the
>> other dyes (eg from the spectral viewer it looks like the 546 will
>> be slightly excited by the 488 laser for AF488, while the red dyes
>> (below) will be slightly excited with 561)
>> - interestingly Molecular Probes include 555 in the some of
their
>> triple label kits - I'm wondering why they have chosen that as 546
>> looks at least comparable on the spectral viewer (obviously more
>> going on - as I hope Mike will reveal ;)

Timothy Feinstein

Re: Whole Cell Dye. .

In reply to this post by Sam's Mail

As I recall (it has been a while and the experiment was not
published), we had decent results by staining canine cells with a
chicken polyclonal raised against dog (Abcam #ab353). See for
instance Biochem J. 364:475. If I had remembered better I would have
noted that Na/K ATPase antibodies can be hit or miss for fluorescence
as well as species-specific.

All the best,

Tim

Timothy Feinstein, PhD
Postdoctoral Associate, Vilardaga laboratory
University of Pittsburgh Dept. of Pharmacology and Chemical Biology
Pittsburgh, PA

On Aug 6, 2009, at 9:57 PM, 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

Marc Thibault

Re: Transwell chambers

In reply to this post by B. Prabhakar Pandian

HI,
In this article we have coated transwell chambers with proteins to promote
haptotaxis (and also tested migration with soluble proteins).

Title: Fibronectin, vitronectin, and collagen I induce chemotaxis and
haptotaxis of human and rabbit mesenchymal stem cells in a standardized
transmembrane assay
Author(s): Thibault MM, Hoemann CD, Buschmann MD
Source: STEM CELLS AND DEVELOPMENT Volume: 16 Issue: 3 Pages: 489-502
Published: JUN 2007

Thanks for asking !
M

-----Original Message-----
From: B. Prabhakar Pandian [mailto:[hidden email]]
Sent: August 6, 2009 7:04 PM
To: [hidden email]
Subject: Transwell chambers

Hello everybody,
Can anyone point to vendors who might be able to
give some samples of the transwell chambers. Also if there is any good
paper that compares all the transwell chambers, that will be great.
We want to see which chambers will be good to coat with extracellular
matrix with different gelatin concentrations and then do TEER measurements.

Thanks,

-Prabhakar

Marc Thibault

Re: Whole Cell Dye

In reply to this post by Cameron Nowell

HI,
CellMasks has worked for us with Hek 293 and hepg2 with rapid post-stain
imaging. Calcein am was also used to stain cell volumes, although it leaks
it is also preferable to image in the first hour post-staining.
M

-----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.

Syed J Khundmiri

Re: Whole Cell Dye. .

In reply to this post by Sam's Mail

We have used Na-K antibodies from Sigma RBI. You can also try the Developmental Hybridoma Bank at Iowa University. The DHSB antibody gave us the best results for IHC and confocal.

Syed

Syed J. Khundmiri, PhD
Assistant Professor Medicine and Physiology
Kidney Disease Program
University of Louisville
570 S. Preston St. Room 102
Louisville, KY 40202
Phone: 502-852-0014
Fax: 502-852-4384
email: [hidden email]
[hidden email]

>>> "Connell, Samuel" <[hidden email]> 8/6/2009 9:57 PM >>>
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

Email Disclaimer: www.stjude.org/emaildisclaimer

Goodhouse, Joseph G.

Re: Whole Cell Dye

In reply to this post by Cameron Nowell

In my experience the best dye we have used is MP's Cell Tracker
Green. It fills the entire cytoplasm of live cells and is nontoxic. In
virally infected cells the staining may be a bit more uneven but on the
whole works very well. We have used it to determine cell volume changes
due to infection.

Joe Goodhouse
Confocal Core Lab Manager
Dept. of Molecular Biology
Princeton University
609-258-5432

Visit us at http://www.molbio1.princeton.edu/facility/confocal/

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Cameron Nowell
Sent: Thursday, August 06, 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.