Nuclear staining of live, intact plant material (roots and leaves)

[Gregg Sobocinski]

We have researchers seeking to stain the nuclei in live plant tissues. DAPI will not penetrate, even in root material. Intentionally tearing or delaminating leaves will allow DAPI penetration for a short distance, but is too invasive to the living cells.

 

Has anyone tried ethidium bromide on whole, plant tissues? How about some of the other nuclear stains?

 

The ultimate goal is double and triple staining with GFP to image on a laser confocal microscope. Fixation would be okay for one of the projects, but evidently GFP fades drastically when fixed.

 

Anyone have experience or suggestions?

 

Much appreciated,

~Gregg (on behalf of others)

 

Gregg Sobocinski

Microscope Imaging Specialist

University of Michigan, MCDB Dept.

Ann Arbor, Michigan

USA

 

[JOEL B. SHEFFIELD]

Have you tried Syto 9, from Invitrogen?

-------------- Original message ---------------
We have researchers seeking to stain the nuclei in live plant
tissues. DAPI will not penetrate, even in root material.
Intentionally tearing or delaminating leaves will allow DAPI
penetration for a short distance, but is too invasive to the living
cells.

Has anyone tried ethidium bromide on whole, plant tissues? How about
some of the other nuclear stains?

The ultimate goal is double and triple staining with GFP to image on
a laser confocal microscope. Fixation would be okay for one of the
projects, but evidently GFP fades drastically when fixed.

Anyone have experience or suggestions?

Much appreciated,
~Gregg (on behalf of others)

Gregg Sobocinski
Microscope Imaging Specialist
University of Michigan, MCDB Dept.
Ann Arbor, Michigan
USA


--
Joel B. Sheffield, Ph.D.
Biology Department, Temple University
1900 North 12th Street
Philadelphia, PA 19122
[hidden email]  
[phone], fax [phone]
http://astro.temple.edu/~jbs

[Rosemary.White]

Depending on the tissue, GFP fluorescence is fine after fixation with
(para)formaldehyde in buffer.  Try it and see if it's OK in your tissues.
You may be able to fix fairly lightly - 1-2% formaldehyde for a few minutes,
just enough to permeabilise the membranes without wrecking internal
structures.

If you're looking at Arabidopsis roots, you should be able to see the nuclei
in the transmitted light channel without staining, unless you're looking at
cells in the stele.  Leaves are a bit more tricky, but you can see them in
Arabidopsis epidermal cells.

Many of the syto dyes stain plant nuclei but they tend to stain quite a bit
of other stuff as well, and also mitochondrial and chloroplast DNA.  They
also seem fairly toxic, but I haven't used them extensively and others may
have had more success with living tissues.  Draq5 works to some extent
though it doesn't seem to penetrate intact plant membranes or walls very
well, but I haven't explored this much.

If you leave tissues in propidium iodide long enough, it will eventually get
into the cells, though their membranes may be a bit compromised.  You can
then image PI fluorescence separately from chlorophyll, and from GFP.

good luck,
cheers,
Rosemary

Dr Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

T 61 2 6246 5475
F 61 2 6246 5334
E [hidden email]


On 18/05/10 7:15 AM, "Joel Sheffield" <[hidden email]> wrote:

> Have you tried Syto 9, from Invitrogen?
>
> -------------- Original message ---------------
> We have researchers seeking to stain the nuclei in live plant
> tissues. DAPI will not penetrate, even in root material.
> Intentionally tearing or delaminating leaves will allow DAPI
> penetration for a short distance, but is too invasive to the living
> cells.
>
> Has anyone tried ethidium bromide on whole, plant tissues? How about
> some of the other nuclear stains?
>
> The ultimate goal is double and triple staining with GFP to image on
> a laser confocal microscope. Fixation would be okay for one of the
> projects, but evidently GFP fades drastically when fixed.
>
> Anyone have experience or suggestions?
>
> Much appreciated,
> ~Gregg (on behalf of others)
>
> Gregg Sobocinski
> Microscope Imaging Specialist
> University of Michigan, MCDB Dept.
> Ann Arbor, Michigan
> USA
>
>
> --
> Joel B. Sheffield, Ph.D.
> Biology Department, Temple University
> 1900 North 12th Street
> Philadelphia, PA 19122
> [hidden email]  
> [phone], fax [phone]
> http://astro.temple.edu/~jbs

[Mario-2]

Gregg,

Have you tried any of the Hoechst stains such Hoechst 33342? Like
DAPI it is a minor groove dsDNA stain but with a higher membrane
permeability and suitable for live cells. In my experience, minor
groove dyes have the best specificity for dsDNA so you don't get
staining of other nucleic acids outside the nucleus. If you don't
care, then the SYTO dyes including 11 and 13, besides 9, might be
useful.

Mario M.

>We have researchers seeking to stain the nuclei in live plant
>tissues. DAPI will not penetrate, even in root material.
>Intentionally tearing or delaminating leaves will allow DAPI
>penetration for a short distance, but is too invasive to the living
>cells.
>
>Has anyone tried ethidium bromide on whole, plant tissues? How about
>some of the other nuclear stains?
>
>The ultimate goal is double and triple staining with GFP to image on
>a laser confocal microscope. Fixation would be okay for one of the
>projects, but evidently GFP fades drastically when fixed.
>
>Anyone have experience or suggestions?
>
>Much appreciated,
>~Gregg (on behalf of others)
>
>Gregg Sobocinski
>Microscope Imaging Specialist
>University of Michigan, MCDB Dept.
>Ann Arbor, Michigan
>USA
>

--
________________________________________________________________________________
Mario M. Moronne, Ph.D.

[hidden email]
[hidden email]

[Gregg Sobocinski]

Mario,
Thanks a lot for the feedback. I think Hoechst is definitely worth a try, based on your recommendation. I appreciate the info about the SYTO dyes as well. This is the kind of information we've been looking for before purchasing any new dyes. I'll pass this along to our researchers.

Thanks to others that have responded as well. Any new feedback is still welcome.

Regards,
~Gregg
Gregg Sobocinski
Microscope Imaging Specialist
University of Michigan, MCDB Dept.
3040 Natural Science (Kraus) Bldg
615-2034




-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Mario
Sent: Tuesday, May 18, 2010 5:24 PM
To: [hidden email]
Subject: Re: Nuclear staining of live, intact plant material (roots and leaves)

Gregg,

Have you tried any of the Hoechst stains such Hoechst 33342? Like
DAPI it is a minor groove dsDNA stain but with a higher membrane
permeability and suitable for live cells. In my experience, minor
groove dyes have the best specificity for dsDNA so you don't get
staining of other nucleic acids outside the nucleus. If you don't
care, then the SYTO dyes including 11 and 13, besides 9, might be
useful.

Mario M.

>We have researchers seeking to stain the nuclei in live plant
>tissues. DAPI will not penetrate, even in root material.
>Intentionally tearing or delaminating leaves will allow DAPI
>penetration for a short distance, but is too invasive to the living
>cells.
>
>Has anyone tried ethidium bromide on whole, plant tissues? How about
>some of the other nuclear stains?
>
>The ultimate goal is double and triple staining with GFP to image on
>a laser confocal microscope. Fixation would be okay for one of the
>projects, but evidently GFP fades drastically when fixed.
>
>Anyone have experience or suggestions?
>
>Much appreciated,
>~Gregg (on behalf of others)
>
>Gregg Sobocinski
>Microscope Imaging Specialist
>University of Michigan, MCDB Dept.
>Ann Arbor, Michigan
>USA
>

--
________________________________________________________________________________
Mario M. Moronne, Ph.D.

[hidden email]
[hidden email]

[Hann Ling Wong]

Dear Gregg,

Perhaps your colleagues may try adding a small amount of detergent
like triton. I used 0.1% triton with DAPI for epidermal onion cells
and Arabidopsis lateral roots. Good luck.


Hann Ling WONG, Ph.D.
Associate Professor
Dept. of Biological Science
Faculty of Science (Perak Campus)
Universiti Tunku Abdul Rahman
Jalan Universiti, Bandar Barat
31900 Kampar, Perak
Malaysia
Email: [hidden email]
         [hidden email]

On Tue, May 18, 2010 at 4:45 AM, Sobocinski, Gregg <[hidden email]> wrote:

> We have researchers seeking to stain the nuclei in live plant tissues. DAPI
> will not penetrate, even in root material. Intentionally tearing or
> delaminating leaves will allow DAPI penetration for a short distance, but is
> too invasive to the living cells.
>
>
>
> Has anyone tried ethidium bromide on whole, plant tissues? How about some of
> the other nuclear stains?
>
>
>
> The ultimate goal is double and triple staining with GFP to image on a laser
> confocal microscope. Fixation would be okay for one of the projects, but
> evidently GFP fades drastically when fixed.
>
>
>
> Anyone have experience or suggestions?
>
>
>
> Much appreciated,
>
> ~Gregg (on behalf of others)
>
>
>
> Gregg Sobocinski
>
> Microscope Imaging Specialist
>
> University of Michigan, MCDB Dept.
>
> Ann Arbor, Michigan
>
> USA
>
>

[Alex Bannigan]

Gregg,
I have been trying to stain DNA in live GFP-expressing arabidopsis roots for a
few years and I haven't found anything really good.
I've tried DAPI, SYTO82 (and several other SYTO dyes), DRAQ9, Ethidium
Bromide, Propidium iodide, SYBR Green, Hoechst and probably others. Most of
them stain the DNA to some extent (at least in roots), but the problem I find is
that the combined stress of the DNA dye, the GFP and the laser tends to make
the cells very sad, causing mitosis (which is what I am trying to watch) to
stop, even in cells that have already started. Adding a mutation into the mix
makes things even worse.
I had some success with SYTO82, but, as Rosemary said, it has an annoying
side-effect of heavily staining the mitochondria as well as the nuclear DNA.
If you find anything that works for you, please post a report - I'd LOVE to hear
about it.
Alex

:::::::::::::::::::::::::::::::::::::::::::::
Alex Bannigan
Director of Microscopy
Biology Department, MSC 7801
James Madison University
800 South Main St
Harrisonburg, VA, 22807, USA
[hidden email]
phone: [phone]
http://csm.jmu.edu/biology/bannigax
::::::::::::::::::::::::::::::::::::::::::::

[Gregg Sobocinski]

Alex,
Thanks so much for the useful report about things you've tried. I will compile the responses from the Listerver community, and will make sure you receive a copy for your reference. I'll try to remember to post whatever works for us, although I cannot say what time frame that will take.

At the very least, you are the voice to console us if we continue to have troubles.

Regards,
~Gregg

Gregg Sobocinski
Microscope Imaging Specialist
University of Michigan, MCDB Dept.
Ann Arbor, Michigan
USA

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Alex Bannigan
Sent: Wednesday, May 19, 2010 10:15 AM
To: [hidden email]
Subject: Re: Nuclear staining of live, intact plant material (roots and leaves)

Gregg,
I have been trying to stain DNA in live GFP-expressing arabidopsis roots for a
few years and I haven't found anything really good.
I've tried DAPI, SYTO82 (and several other SYTO dyes), DRAQ9, Ethidium
Bromide, Propidium iodide, SYBR Green, Hoechst and probably others. Most of
them stain the DNA to some extent (at least in roots), but the problem I find is
that the combined stress of the DNA dye, the GFP and the laser tends to make
the cells very sad, causing mitosis (which is what I am trying to watch) to
stop, even in cells that have already started. Adding a mutation into the mix
makes things even worse.
I had some success with SYTO82, but, as Rosemary said, it has an annoying
side-effect of heavily staining the mitochondria as well as the nuclear DNA.
If you find anything that works for you, please post a report - I'd LOVE to hear
about it.
Alex

:::::::::::::::::::::::::::::::::::::::::::::
Alex Bannigan
Director of Microscopy
Biology Department, MSC 7801
James Madison University
800 South Main St
Harrisonburg, VA, 22807, USA
[hidden email]
phone: [phone]
http://csm.jmu.edu/biology/bannigax
::::::::::::::::::::::::::::::::::::::::::::

[Ignatius, Mike-2]

Hello All,

To add to this ever growing offering, we have read reports using SYTO 42, SYBR Green I, and Hoechst 33258 in live plants. If you reply directly to me I can send along an 11 page Plant Technology article one of our Tech Specialist, Dr. Ed Leber, created last year summmarizing over 75 publications using our dyes for plant studies.  From cell walls to nuclei.

We should also mention the new BrdU replacement, Click-iT EdU will label dividing plant cells very well.  They are labeled alive, but detected after fix and perm.  The benefit of EdU is no treatment necessary to expose EdU handle to its detection reagent, unlike the harsh chemical or DNAse treatments required for BrdU.  Shorter too, for most protocols, 7 hrs vs 21hrs.  Detection is any range of colors.

Regards,

Mike Ignatius

Molecular Probes/Life Technologies

[hidden email]


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sobocinski, Gregg
Sent: Wednesday, May 19, 2010 7:30 AM
To: [hidden email]
Subject: Re: Nuclear staining of live, intact plant material (roots and leaves)

Alex,
Thanks so much for the useful report about things you've tried. I will compile the responses from the Listerver community, and will make sure you receive a copy for your reference. I'll try to remember to post whatever works for us, although I cannot say what time frame that will take.

At the very least, you are the voice to console us if we continue to have troubles.

Regards,
~Gregg

Gregg Sobocinski
Microscope Imaging Specialist
University of Michigan, MCDB Dept.
Ann Arbor, Michigan
USA

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Alex Bannigan
Sent: Wednesday, May 19, 2010 10:15 AM
To: [hidden email]
Subject: Re: Nuclear staining of live, intact plant material (roots and leaves)

Gregg,
I have been trying to stain DNA in live GFP-expressing arabidopsis roots for a few years and I haven't found anything really good.
I've tried DAPI, SYTO82 (and several other SYTO dyes), DRAQ9, Ethidium Bromide, Propidium iodide, SYBR Green, Hoechst and probably others. Most of them stain the DNA to some extent (at least in roots), but the problem I find is that the combined stress of the DNA dye, the GFP and the laser tends to make the cells very sad, causing mitosis (which is what I am trying to watch) to stop, even in cells that have already started. Adding a mutation into the mix makes things even worse.
I had some success with SYTO82, but, as Rosemary said, it has an annoying side-effect of heavily staining the mitochondria as well as the nuclear DNA.
If you find anything that works for you, please post a report - I'd LOVE to hear about it.
Alex

:::::::::::::::::::::::::::::::::::::::::::::
Alex Bannigan
Director of Microscopy
Biology Department, MSC 7801
James Madison University
800 South Main St
Harrisonburg, VA, 22807, USA
[hidden email]
phone: [phone]
http://csm.jmu.edu/biology/bannigax
::::::::::::::::::::::::::::::::::::::::::::

[Rosemary.White]

Dear all,

Sorry for delayed reply, email crash hid this in another folder....

The main thing to remember is that SOME of these DNA dyes will work
fantastically for SOME live plant tissues.  DAPI is a fabulous stain for DNA
in live nuclei in many algae for example.  It depends on a combination of
the cell wall and membrane. You just have to try them out, but it can get
jolly expensive.  

And almost all DNA stains, at least in my hands, stain the plant cell wall,
which is sometimes a pain.  The common groove-binders like DAPI, Hoechst and
PI certainly do, and if I remember correctly, the syto dyes do, and so does
DRAQ5.

The membrane properties - types of phospholipid, size of membrane potential,
etc. will affect uptake of these charged dyes.  Just had a thought that you
may be able to permeabilise with ionophores (rather than detergents) to
allow dye entry.  Haven't tried it but might be worth a go.

In the long run, if you're working with Arabidopsis and want to see live
nuclei, it may be worthwhile to cross your GFP-tagged line with a line
expressing mTomato- or citrine- or cerulean-tagged histone or perhaps a
tagged nuclear envelope protein (that isn't also in the ER).  There's
usually associated antibiotic resistance you can use to get homozygous
lines.  For chromosomes/DNA, it may be possible to tag a DNA-binding protein
or some other nuclear DNA-associated protein - I don't know if such lines
are available from the Arabidopsis stock centre.  Would be worth asking the
Arabidopsis list if interested.

cheers,
Rosemary

On 20/05/10 1:56 AM, "Ignatius, Mike" <[hidden email]> wrote:

> Hello All,
>
> To add to this ever growing offering, we have read reports using SYTO 42, SYBR
> Green I, and Hoechst 33258 in live plants. If you reply directly to me I can
> send along an 11 page Plant Technology article one of our Tech Specialist, Dr.
> Ed Leber, created last year summmarizing over 75 publications using our dyes
> for plant studies.  From cell walls to nuclei.
>
> We should also mention the new BrdU replacement, Click-iT EdU will label
> dividing plant cells very well.  They are labeled alive, but detected after
> fix and perm.  The benefit of EdU is no treatment necessary to expose EdU
> handle to its detection reagent, unlike the harsh chemical or DNAse treatments
> required for BrdU.  Shorter too, for most protocols, 7 hrs vs 21hrs.
> Detection is any range of colors.
>
> Regards,
>
> Mike Ignatius
>
> Molecular Probes/Life Technologies
>
> [hidden email]
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On
> Behalf Of Sobocinski, Gregg
> Sent: Wednesday, May 19, 2010 7:30 AM
> To: [hidden email]
> Subject: Re: Nuclear staining of live, intact plant material (roots and
> leaves)
>
> Alex,
> Thanks so much for the useful report about things you've tried. I will compile
> the responses from the Listerver community, and will make sure you receive a
> copy for your reference. I'll try to remember to post whatever works for us,
> although I cannot say what time frame that will take.
>
> At the very least, you are the voice to console us if we continue to have
> troubles.
>
> Regards,
> ~Gregg
>
> Gregg Sobocinski
> Microscope Imaging Specialist
> University of Michigan, MCDB Dept.
> Ann Arbor, Michigan
> USA
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On
> Behalf Of Alex Bannigan
> Sent: Wednesday, May 19, 2010 10:15 AM
> To: [hidden email]
> Subject: Re: Nuclear staining of live, intact plant material (roots and
> leaves)
>
> Gregg,
> I have been trying to stain DNA in live GFP-expressing arabidopsis roots for a
> few years and I haven't found anything really good.
> I've tried DAPI, SYTO82 (and several other SYTO dyes), DRAQ9, Ethidium
> Bromide, Propidium iodide, SYBR Green, Hoechst and probably others. Most of
> them stain the DNA to some extent (at least in roots), but the problem I find
> is that the combined stress of the DNA dye, the GFP and the laser tends to
> make the cells very sad, causing mitosis (which is what I am trying to watch)
> to stop, even in cells that have already started. Adding a mutation into the
> mix makes things even worse.
> I had some success with SYTO82, but, as Rosemary said, it has an annoying
> side-effect of heavily staining the mitochondria as well as the nuclear DNA.
> If you find anything that works for you, please post a report - I'd LOVE to
> hear about it.
> Alex
>
> :::::::::::::::::::::::::::::::::::::::::::::
> Alex Bannigan
> Director of Microscopy
> Biology Department, MSC 7801
> James Madison University
> 800 South Main St
> Harrisonburg, VA, 22807, USA
> [hidden email]
> phone: [phone]
> http://csm.jmu.edu/biology/bannigax
> ::::::::::::::::::::::::::::::::::::::::::::

[Zucker.Robert]

Mike
can you send me this article on plant stains. It may provide the
necessary material and knowledge for my next Nikon submission
what did Leica stain there famous plant cell with that they use for QA.
bob





Robert Zucker PhD
USEPA NHEERL TAD
MD 67
RTP NC 27711

Tel: [phone]
fax -919-541-4017

shipping address

RTF building
2525 E.NC highway 54
Durham NC 27713


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  >----------------------------------------------------------------------------------------------------------------------------------------|





Hello All,

To add to this ever growing offering, we have read reports using SYTO
42, SYBR Green I, and Hoechst 33258 in live plants. If you reply
directly to me I can send along an 11 page Plant Technology article one
of our Tech Specialist, Dr. Ed Leber, created last year summmarizing
over 75 publications using our dyes for plant studies.  From cell walls
to nuclei.

We should also mention the new BrdU replacement, Click-iT EdU will label
dividing plant cells very well.  They are labeled alive, but detected
after fix and perm.  The benefit of EdU is no treatment necessary to
expose EdU handle to its detection reagent, unlike the harsh chemical or
DNAse treatments required for BrdU.  Shorter too, for most protocols, 7
hrs vs 21hrs.  Detection is any range of colors.

Regards,

Mike Ignatius

Molecular Probes/Life Technologies

[hidden email]


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Sobocinski, Gregg
Sent: Wednesday, May 19, 2010 7:30 AM
To: [hidden email]
Subject: Re: Nuclear staining of live, intact plant material (roots and
leaves)

Alex,
Thanks so much for the useful report about things you've tried. I will
compile the responses from the Listerver community, and will make sure
you receive a copy for your reference. I'll try to remember to post
whatever works for us, although I cannot say what time frame that will
take.

At the very least, you are the voice to console us if we continue to
have troubles.

Regards,
~Gregg

Gregg Sobocinski
Microscope Imaging Specialist
University of Michigan, MCDB Dept.
Ann Arbor, Michigan
USA

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Alex Bannigan
Sent: Wednesday, May 19, 2010 10:15 AM
To: [hidden email]
Subject: Re: Nuclear staining of live, intact plant material (roots and
leaves)

Gregg,
I have been trying to stain DNA in live GFP-expressing arabidopsis roots
for a few years and I haven't found anything really good.
I've tried DAPI, SYTO82 (and several other SYTO dyes), DRAQ9, Ethidium
Bromide, Propidium iodide, SYBR Green, Hoechst and probably others. Most
of them stain the DNA to some extent (at least in roots), but the
problem I find is that the combined stress of the DNA dye, the GFP and
the laser tends to make the cells very sad, causing mitosis (which is
what I am trying to watch) to stop, even in cells that have already
started. Adding a mutation into the mix makes things even worse.
I had some success with SYTO82, but, as Rosemary said, it has an
annoying side-effect of heavily staining the mitochondria as well as the
nuclear DNA.
If you find anything that works for you, please post a report - I'd LOVE
to hear about it.
Alex

:::::::::::::::::::::::::::::::::::::::::::::
Alex Bannigan
Director of Microscopy
Biology Department, MSC 7801
James Madison University
800 South Main St
Harrisonburg, VA, 22807, USA
[hidden email]
phone: [phone]
http://csm.jmu.edu/biology/bannigax
::::::::::::::::::::::::::::::::::::::::::::

[mersorica]

Hello,

thanks for all the info, could you send me the pdf?

Regards