Phalloidin background on transwell membrane

[Renato A. Mortara]

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Dear all
 
A colleague of mine is trying to label polarized epithelial cells grown on
transwell dishes.
 
However, fluorescent phalloidin is labeling the dish membrane, rendering it
impossible to image the cells.
 
She tried to block with goat serum, BSA with no luck.
 
Any suggestions ?
 
Many thanks !
 
Renato
 
Renato A. Mortara
Parasitology Division
UNIFESP - Escola Paulista de Medicina
Rua Botucatu, 862, 6th floor
São Paulo, SP
04023-062
Brazil
Phone: [phone]
Fax:     [phone]
email: [hidden email]
home page: www.ecb.epm.br/~ramortara

[Tobias Baskin]

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Try non fluorescent phalloidin??

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>To join, leave or search the confocal microscopy listserv, go to:
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>
>Dear all
>
>A colleague of mine is trying to label polarized epithelial cells grown on
>transwell dishes.
>
>However, fluorescent phalloidin is labeling the dish membrane, rendering it
>impossible to image the cells.
>
>She tried to block with goat serum, BSA with no luck.
>
>Any suggestions ?
>
>Many thanks !
>
>Renato
>
>Renato A. Mortara
>Parasitology Division
>UNIFESP - Escola Paulista de Medicina
>Rua Botucatu, 862, 6th floor
>São Paulo, SP
>04023-062
>Brazil
>Phone: [phone]
>Fax:     [phone]
>email: [hidden email]
>home page: www.ecb.epm.br/~ramortara

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[Ryan Schreiner]

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We use polycarbonate transwells for fluorescent studies and do not
have problems with filter background, whereas the many of the clear
filters can have increased background signal, the CF series of
fluorophores coupled to phalloidin from Biotium are particularly good.

On Tue, Jun 14, 2011 at 10:30 AM, Renato Mortara <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all
>
> A colleague of mine is trying to label polarized epithelial cells grown on
> transwell dishes.
>
> However, fluorescent phalloidin is labeling the dish membrane, rendering it
> impossible to image the cells.
>
> She tried to block with goat serum, BSA with no luck.
>
> Any suggestions ?
>
> Many thanks !
>
> Renato
>
> Renato A. Mortara
> Parasitology Division
> UNIFESP - Escola Paulista de Medicina
> Rua Botucatu, 862, 6th floor
> São Paulo, SP
> 04023-062
> Brazil
> Phone: [phone]
> Fax:     [phone]
> email: [hidden email]
> home page: www.ecb.epm.br/~ramortara
>

[Glen MacDonald-2]

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What is the fluorophore?  I found that mitotracker red tends to stick to the Millipore membranes but MT green does not.  Red fluorophores tend to be more lipophilic.  
The Probes ImageiT fx might help block the sticking.

Regards,
Glen

Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923
[phone]

On Tue, 14 Jun 2011, Renato Mortara wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all
>
> A colleague of mine is trying to label polarized epithelial cells grown on
> transwell dishes.
>
> However, fluorescent phalloidin is labeling the dish membrane, rendering it
> impossible to image the cells.
>
> She tried to block with goat serum, BSA with no luck.
>
> Any suggestions ?
>
> Many thanks !
>
> Renato
>
> Renato A. Mortara
> Parasitology Division
> UNIFESP - Escola Paulista de Medicina
> Rua Botucatu, 862, 6th floor
> São Paulo, SP
> 04023-062
> Brazil
> Phone: [phone]
> Fax:     [phone]
> email: [hidden email]
> home page: www.ecb.epm.br/~ramortara
>

[Cameron, Lisa]

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I would recommend looking at the dish membrane without any fluorophore labeling
to check for autofluorescence. I've had a couple people use membranes that are
autofluorescent - mostly in the 488nm range.
So red or far red phalloidin would be better in this case, perhaps.
- Lisa


---------------------------------------
Lisa Cameron, Ph.D.
Director of Confocal and Light Microscopy Core
Dana Farber Cancer Institute
Boston, MA 02215
[hidden email]


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Renato Mortara
Sent: Tuesday, June 14, 2011 10:30 AM
To: [hidden email]
Subject: [CONFOCALMICROSCOPY] Phalloidin background on transwell membrane

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Dear all
 
A colleague of mine is trying to label polarized epithelial cells grown on
transwell dishes.
 
However, fluorescent phalloidin is labeling the dish membrane, rendering it
impossible to image the cells.
 
She tried to block with goat serum, BSA with no luck.
 
Any suggestions ?
 
Many thanks !
 
Renato
 
Renato A. Mortara
Parasitology Division
UNIFESP - Escola Paulista de Medicina
Rua Botucatu, 862, 6th floor
São Paulo, SP
04023-062
Brazil
Phone: [phone]
Fax:     [phone]
email: [hidden email]
home page: www.ecb.epm.br/~ramortara


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[dlevine]

Or you can try a transwell-like device that does not bind dye or autofluoresce.  CytoVu from SiMPore uses a Silicon membrane that is perfect for fluorescent microscopy.  Check it out at SiMPoreStore.com

Daniel Levine

[George McNamara]

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use fluorescent anti-actin


On 6/14/2011 10:30 AM, Renato Mortara wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all
>
> A colleague of mine is trying to label polarized epithelial cells grown on
> transwell dishes.
>
> However, fluorescent phalloidin is labeling the dish membrane, rendering it
> impossible to image the cells.
>
> She tried to block with goat serum, BSA with no luck.
>
> Any suggestions ?
>
> Many thanks !
>
> Renato
>
> Renato A. Mortara
> Parasitology Division
> UNIFESP - Escola Paulista de Medicina
> Rua Botucatu, 862, 6th floor
> São Paulo, SP
> 04023-062
> Brazil
> Phone: [phone]
> Fax:     [phone]
> email: [hidden email]
> home page: www.ecb.epm.br/~ramortara
>
>    

--


George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

[Julian Smith III]

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If you're using Alexas or Cys, check that the transwell membrane doesn't
have a huge positive charge on it.  If so, try an overnight rinse in
buffer with 1% BSA. That trick works to compete charge-bonded dye off of
strongly positive (basic) protein secretion bodies in our worm wholemounts.
Julian

On 6/15/11 9:31 PM, George McNamara wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> use fluorescent anti-actin
>
>
> On 6/14/2011 10:30 AM, Renato Mortara wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear all
>>
>> A colleague of mine is trying to label polarized epithelial cells
>> grown on
>> transwell dishes.
>>
>> However, fluorescent phalloidin is labeling the dish membrane,
>> rendering it
>> impossible to image the cells.
>>
>> She tried to block with goat serum, BSA with no luck.
>>
>> Any suggestions ?
>>
>> Many thanks !
>>
>> Renato
>>
>> Renato A. Mortara
>> Parasitology Division
>> UNIFESP - Escola Paulista de Medicina
>> Rua Botucatu, 862, 6th floor
>> São Paulo, SP
>> 04023-062
>> Brazil
>> Phone: [phone]
>> Fax:     [phone]
>> email: [hidden email]
>> home page: www.ecb.epm.br/~ramortara
>>
>
>

--
Julian P.S. Smith III
Director, Winthrop Microscopy Facility
Dept. of Biology
Winthrop University
520 Cherry Rd.
Rock Hill, SC  29733

803-323-2111 x6427 (vox)
803-323-3448 (fax)
803-524-2347 (cell)
Research Website www.birdnest.org/smithj
Personal Website www.rociada-east.net