stable phalloidin labeling?

[Harshad Vishwasrao]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Greetings from a first time poster,

I'm staining f-actin in fixed mammalian cell cultures using fluorescent phalloidin.
What I've noticed is that the staining typically works very well and lasts for a
few days without much change. Then I typically lose the phalloidin stain very
quickly (overnight) and re-staining does not get the pattern back - which
possibly indicates the structure of the f-actin itself is degrading. This process
is accelerated if I do more manipulations to my sample (other stains, washes,
etc.).

The fixation/permeablization/staining protocol I use is pretty standard. 15 min
4% PFA fix. 10 min 0.3% TritonX permeablization. 20min Invitrogen Phalloidin-
Alexa488 or Actistain488 stain (or other fluorophore). PBS is the base media
throughout. I do multiple manipulations (other stains, etc) to my sample over
the course of several days after fixation, so I can't mount and seal my samples.  

I wanted to ask if anyone has seen something like this sudden and irreversible
phalloidin loss and whether you've found a solution to keep phalloidin stably
bound.

Thanks very much in advance,
Harsh

[mcammer]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Try fixing 20 min?  After staining add fix?  Try a different batch of phalloidin?

We've seen this happen every once in a while and have never figured out why.  Most likely scenario, I think, is the last-- the dye falls off, but really don't know.  The lot we have of Alexa 488 Phalloidin in the freezer has stained cells for more than two years that stay bright for months.  

===========================================================================
Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
Cell:  914-309-3270   note that we do not receive messages left at 212-263-3208
http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Harshad Vishwasrao
Sent: Thursday, November 13, 2014 2:26 PM
To: [hidden email]
Subject: stable phalloidin labeling?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Greetings from a first time poster,

I'm staining f-actin in fixed mammalian cell cultures using fluorescent phalloidin.
What I've noticed is that the staining typically works very well and lasts for a few days without much change. Then I typically lose the phalloidin stain very quickly (overnight) and re-staining does not get the pattern back - which possibly indicates the structure of the f-actin itself is degrading. This process is accelerated if I do more manipulations to my sample (other stains, washes, etc.).

The fixation/permeablization/staining protocol I use is pretty standard. 15 min 4% PFA fix. 10 min 0.3% TritonX permeablization. 20min Invitrogen Phalloidin-
Alexa488 or Actistain488 stain (or other fluorophore). PBS is the base media throughout. I do multiple manipulations (other stains, etc) to my sample over the course of several days after fixation, so I can't mount and seal my samples.  

I wanted to ask if anyone has seen something like this sudden and irreversible phalloidin loss and whether you've found a solution to keep phalloidin stably bound.

Thanks very much in advance,
Harsh

[David Crossman]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

I have had problems with phalloidin on fixed cardiac tissue when the sections were mounted in citifluor antifade. The labelling would dissociate overnight. Perhaps this could be an explanation?

Cheers,
David  

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cammer, Michael
Sent: Friday, 14 November 2014 8:48 a.m.
To: [hidden email]
Subject: Re: stable phalloidin labeling?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Try fixing 20 min?  After staining add fix?  Try a different batch of phalloidin?

We've seen this happen every once in a while and have never figured out why.  Most likely scenario, I think, is the last-- the dye falls off, but really don't know.  The lot we have of Alexa 488 Phalloidin in the freezer has stained cells for more than two years that stay bright for months.  

===========================================================================
Michael Cammer, Microscopy Core & Skirball Institute, NYU Langone Medical Center
Cell:  914-309-3270   note that we do not receive messages left at 212-263-3208
http://ocs.med.nyu.edu/microscopy & http://microscopynotes.com/


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Harshad Vishwasrao
Sent: Thursday, November 13, 2014 2:26 PM
To: [hidden email]
Subject: stable phalloidin labeling?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Greetings from a first time poster,

I'm staining f-actin in fixed mammalian cell cultures using fluorescent phalloidin.
What I've noticed is that the staining typically works very well and lasts for a few days without much change. Then I typically lose the phalloidin stain very quickly (overnight) and re-staining does not get the pattern back - which possibly indicates the structure of the f-actin itself is degrading. This process is accelerated if I do more manipulations to my sample (other stains, washes, etc.).

The fixation/permeablization/staining protocol I use is pretty standard. 15 min 4% PFA fix. 10 min 0.3% TritonX permeablization. 20min Invitrogen Phalloidin-
Alexa488 or Actistain488 stain (or other fluorophore). PBS is the base media throughout. I do multiple manipulations (other stains, etc) to my sample over the course of several days after fixation, so I can't mount and seal my samples.  

I wanted to ask if anyone has seen something like this sudden and irreversible phalloidin loss and whether you've found a solution to keep phalloidin stably bound.

Thanks very much in advance,
Harsh

[Eric Griffis]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Hi Harsh,

I find that a double fixation helps with maintaining actin structure. We will do an initial fix for 1 minute with 4% PFA, then permeabilize for 5 min with 0.5% TX-100, and then fix again for 10 minutes with 4% PFA. We also use PHEM (PIPES, HEPES, EGTA, MgCl2) instead of PBS for diluting PFA and Triton. You could also try a simultaneous fixation/permeabilization. I like 0.3 % Glutaraldehyde, 4% PFA (Freshly made) in PHEM supplemented with saponin (1mg/ml) for 10 minutes. Then I permeabliize cells with PHEM+0.5% TX-100 for 5 minutes, rinse cells with 3x in PHEM, and then reduce unreacted aldehydes in the sample by treatment for 10 minutes with a solution of 1 mg/mL sodium borohydride dissolved in PHEM (Make immediately before use), and I repeat this twice. I then wash 4x with PHEM+0.1% TX-100 and go on with my blocking and staining. I also find that adding the phalloidin in with my secondary antibodies works well.

We also found that certain mounting agents are better than others for maintaining phalloidin staining. Dako (no commercial interest) always worked well for us.

Hope this is helpful.

Eric




Centre for Gene Regulation and Expression
College of Life Sciences
University of Dundee
MSI/WTB/JBC Complex
Dow Street
Dundee DD1 5EH
United KIngdom

[phone]
[hidden email]<mailto:[hidden email]>
http://www.lifesci.dundee.ac.uk/groups/eric_griffis/GriffisLab/index.html



On 13 Nov 2014, at 19:25, Harshad Vishwasrao <[hidden email]<mailto:[hidden email]>>
 wrote:

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Greetings from a first time poster,

I'm staining f-actin in fixed mammalian cell cultures using fluorescent phalloidin.
What I've noticed is that the staining typically works very well and lasts for a
few days without much change. Then I typically lose the phalloidin stain very
quickly (overnight) and re-staining does not get the pattern back - which
possibly indicates the structure of the f-actin itself is degrading. This process
is accelerated if I do more manipulations to my sample (other stains, washes,
etc.).

The fixation/permeablization/staining protocol I use is pretty standard. 15 min
4% PFA fix. 10 min 0.3% TritonX permeablization. 20min Invitrogen Phalloidin-
Alexa488 or Actistain488 stain (or other fluorophore). PBS is the base media
throughout. I do multiple manipulations (other stains, etc) to my sample over
the course of several days after fixation, so I can't mount and seal my samples.

I wanted to ask if anyone has seen something like this sudden and irreversible
phalloidin loss and whether you've found a solution to keep phalloidin stably
bound.

Thanks very much in advance,
Harsh


The University of Dundee is a registered Scottish Charity, No: SC015096