Immuno-labelling leaf material. Please help!

[Shane van Breda]

I am currently trying to determine the tissue and intracellular localisation of
caffeine in C.sinensis (tea) leaves. I have a polyclonal caffeine antibody Ig
fraction that is directly labelled to FTC-ED.

I am struggling to find literature of labelling leaves with flourescently labelled
antibodies. Would it be possible to label fresh material without fixing or
dehydration (I am worried these steps will dissolve or remove caffeine from
the leaf).

Alternatively I will be doing high pressure freezing and freeze substitution (in a
solvent that caffeine does not dissovle in) or maybe freeze drying in 3 weeks
to embedd my samples. But how would I ensure labelling with my antibody?
and will I still need to premeablise my cells?

Another idea I have is to determine if caffeine has any type of
autoflourescence and then use this to aid in visualising it.

Any ideas, references or protocols that could give me a starting point will be
much appreciated!

[Guy Cox-2]

I have immuno-labelled leaf epidermal peels.  I found that mild
paraformaldehyde fixation and cellulase treatment were necessary to get
penetration.  I can't answer for what that will do for caffeine, since I
don't know how easy it is to leach (though we do use boiling water to
make coffee or tea) but short of microinjection I can't see what would
do better.  Anything that would allow the antibody to enter would surely
allow caffeine to dissolve, it's just a question of how fast it
dissolves at room temperature.  Another point - leaf tissue after this
sort of treatment is not robust, so I found it was essential to fix the
epidermis to coverslips with poly-l-lysine.  Otherwise, even if they
looked OK, the intracellular structure was scrambled.

                                           Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Shane van Breda
Sent: Monday, 31 May 2010 7:18 PM
To: [hidden email]
Subject: Immuno-labelling leaf material. Please help!

I am currently trying to determine the tissue and intracellular
localisation of
caffeine in C.sinensis (tea) leaves. I have a polyclonal caffeine
antibody Ig
fraction that is directly labelled to FTC-ED.

I am struggling to find literature of labelling leaves with
flourescently labelled
antibodies. Would it be possible to label fresh material without fixing
or
dehydration (I am worried these steps will dissolve or remove caffeine
from
the leaf).

Alternatively I will be doing high pressure freezing and freeze
substitution (in a
solvent that caffeine does not dissovle in) or maybe freeze drying in 3
weeks
to embedd my samples. But how would I ensure labelling with my antibody?

and will I still need to premeablise my cells?

Another idea I have is to determine if caffeine has any type of
autoflourescence and then use this to aid in visualising it.

Any ideas, references or protocols that could give me a starting point
will be
much appreciated!

[Tobias Baskin]

Shane,
        Unless caffeine is stored in some kind of small vesicle, it
is unlikely that any kind of aqueous procedure that gives access to
your antibody will also allow all the caffeine to be lost.

        You mentioned cryofixation and freeze substitution. This has
been used before to localize small soluble molecules. Of course,
there is no guarantee it will work. After substitution, you
infiltrate into the resin of your choice, embed, and section. Then
stain the sections.

        Note that if you need to localize only at the level of light
microscope, then you can  embed in butylmethylmetacrylate. THis resin
is not so great for TEM but is very nice for immuowork. It is easy to
make and section, and it can be removed with a brief acetone exposure
after sectioning, which gives great access of the structure to your
antibody. E-mail me off line if you want to pursue this resin further.

        While I don't know that anyone has gone after caffeine, other
small molecules have been chased around. If you get into the
literature on freeze substitution you can find some papers on folks
who have localized small molecules.

        The idea of detecting the caffeine spectrally is interesting
and that might work well on sectioned material with some kind of
microspectoscropic approach, particularly if the caffeine is in the
vacoule.

        Yours is no easy problem! Good luck!
                                Tobias

At 4:18 AM -0500 5/31/10, Shane van Breda wrote:

>I am currently trying to determine the tissue and intracellular
>localisation of
>caffeine in C.sinensis (tea) leaves. I have a polyclonal caffeine antibody Ig
>fraction that is directly labelled to FTC-ED.
>
>I am struggling to find literature of labelling leaves with
>flourescently labelled
>antibodies. Would it be possible to label fresh material without fixing or
>dehydration (I am worried these steps will dissolve or remove caffeine from
>the leaf).
>
>Alternatively I will be doing high pressure freezing and freeze
>substitution (in a
>solvent that caffeine does not dissovle in) or maybe freeze drying in 3 weeks
>to embedd my samples. But how would I ensure labelling with my antibody?
>and will I still need to premeablise my cells?
>
>Another idea I have is to determine if caffeine has any type of
>autoflourescence and then use this to aid in visualising it.
>
>Any ideas, references or protocols that could give me a starting point will be
>much appreciated!

--
       _      ____          __   ____  
      /  \   /          / \    /   \ \        Tobias I. Baskin
     /   /  /          /   \   \      \         Biology Department
    /_ /   __      /__ \   \       \__    611 N. Pleasant St.
   /      /          /       \   \       \        University of Massachusetts
  /      /          /         \   \       \    Amherst, MA, 01003
/      / ___   /           \   \__/  \ ____
www.bio.umass.edu/biology/baskin
Voice: 413 - 545 - 1533 Fax: 413 - 545 - 3243

[Martin Wessendorf-2]

Dear Guy et al--

Never having done a bit of work in plants, I have no idea of the
considerations involved here.  However, based on the chemistry of
caffeine--i.e., it's soluble in polar solvents and has no free primary
amines or carboxyl groups--it seems as if it'd be unlikely to stay in
place without fixation but hard to fix covalently.  The only way I know
that might fix the stuff would be with McLean-Nakane fixation, where the
ketones would get oxidized to aldehydes, but that would probably render
it unrecognizable by the antibody.

Is there any way that polarized microscopy could be used, since caffeine
is birefringent?  I realize that starch is also birefringent, but is
there likely to be much starch in tea leaves?

Martin

Guy Cox wrote:

> I have immuno-labelled leaf epidermal peels.  I found that mild
> paraformaldehyde fixation and cellulase treatment were necessary to get
> penetration.  I can't answer for what that will do for caffeine, since I
> don't know how easy it is to leach (though we do use boiling water to
> make coffee or tea) but short of microinjection I can't see what would
> do better.  Anything that would allow the antibody to enter would surely
> allow caffeine to dissolve, it's just a question of how fast it
> dissolves at room temperature.  Another point - leaf tissue after this
> sort of treatment is not robust, so I found it was essential to fix the
> epidermis to coverslips with poly-l-lysine.  Otherwise, even if they
> looked OK, the intracellular structure was scrambled.
>
>                                            Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>      http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>              Mobile [phone]
> ______________________________________________
>       http://www.guycox.net
>  
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Shane van Breda
> Sent: Monday, 31 May 2010 7:18 PM
> To: [hidden email]
> Subject: Immuno-labelling leaf material. Please help!
>
> I am currently trying to determine the tissue and intracellular
> localisation of
> caffeine in C.sinensis (tea) leaves. I have a polyclonal caffeine
> antibody Ig
> fraction that is directly labelled to FTC-ED.
>
> I am struggling to find literature of labelling leaves with
> flourescently labelled
> antibodies. Would it be possible to label fresh material without fixing
> or
> dehydration (I am worried these steps will dissolve or remove caffeine
> from
> the leaf).
>
> Alternatively I will be doing high pressure freezing and freeze
> substitution (in a
> solvent that caffeine does not dissovle in) or maybe freeze drying in 3
> weeks
> to embedd my samples. But how would I ensure labelling with my antibody?
>
> and will I still need to premeablise my cells?
>
> Another idea I have is to determine if caffeine has any type of
> autoflourescence and then use this to aid in visualising it.
>
> Any ideas, references or protocols that could give me a starting point
> will be
> much appreciated!

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Guy Cox-2]

Yes, there will be starch in tea leaves, but it will be recognizably
confined to the chloroplasts so if caffeine is in crystalline granules
they could be recognizable.  However, the cellulose of the cell walls is
also highly birefringent and this would create a pretty high background.
And the caffeine may well be in solution even if it is in discrete
vesicles.  This is a tricky one - would caffeine have a recognizable
signature in Raman microscopy?

                                           Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Martin Wessendorf
Sent: Wednesday, 2 June 2010 1:32 AM
To: [hidden email]
Subject: Re: Immuno-labelling leaf material. Please help!

Dear Guy et al--

Never having done a bit of work in plants, I have no idea of the
considerations involved here.  However, based on the chemistry of
caffeine--i.e., it's soluble in polar solvents and has no free primary
amines or carboxyl groups--it seems as if it'd be unlikely to stay in
place without fixation but hard to fix covalently.  The only way I know
that might fix the stuff would be with McLean-Nakane fixation, where the

ketones would get oxidized to aldehydes, but that would probably render
it unrecognizable by the antibody.

Is there any way that polarized microscopy could be used, since caffeine

is birefringent?  I realize that starch is also birefringent, but is
there likely to be much starch in tea leaves?

Martin

Guy Cox wrote:
> I have immuno-labelled leaf epidermal peels.  I found that mild
> paraformaldehyde fixation and cellulase treatment were necessary to
get
> penetration.  I can't answer for what that will do for caffeine, since
I
> don't know how easy it is to leach (though we do use boiling water to
> make coffee or tea) but short of microinjection I can't see what would
> do better.  Anything that would allow the antibody to enter would
surely
> allow caffeine to dissolve, it's just a question of how fast it
> dissolves at room temperature.  Another point - leaf tissue after this
> sort of treatment is not robust, so I found it was essential to fix
the

> epidermis to coverslips with poly-l-lysine.  Otherwise, even if they
> looked OK, the intracellular structure was scrambled.
>
>                                            Guy
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>      http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
>
> Phone [phone]     Fax [phone]
>              Mobile [phone]
> ______________________________________________
>       http://www.guycox.net
>  
>
>
> -----Original Message-----
> From: Confocal Microscopy List

[mailto:[hidden email]]

> On Behalf Of Shane van Breda
> Sent: Monday, 31 May 2010 7:18 PM
> To: [hidden email]
> Subject: Immuno-labelling leaf material. Please help!
>
> I am currently trying to determine the tissue and intracellular
> localisation of
> caffeine in C.sinensis (tea) leaves. I have a polyclonal caffeine
> antibody Ig
> fraction that is directly labelled to FTC-ED.
>
> I am struggling to find literature of labelling leaves with
> flourescently labelled
> antibodies. Would it be possible to label fresh material without

fixing
> or
> dehydration (I am worried these steps will dissolve or remove caffeine
> from
> the leaf).
>
> Alternatively I will be doing high pressure freezing and freeze
> substitution (in a
> solvent that caffeine does not dissovle in) or maybe freeze drying in
3
> weeks
> to embedd my samples. But how would I ensure labelling with my
antibody?
>
> and will I still need to premeablise my cells?
>
> Another idea I have is to determine if caffeine has any type of
> autoflourescence and then use this to aid in visualising it.
>
> Any ideas, references or protocols that could give me a starting point
> will be
> much appreciated!

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Shane van Breda]

Thanks to everyone for their help on my topic!

The fact that caffeine is birefringent is very interesting and I will be looking
into this. In our on HPLC analysis it seems that caffeine is present in quite a
high concentration compared to other alkaloids and starch.

One problem that exists is the possibility that caffeine is synthesized in the
chloroplasts (caffeine synthase [responsible for caffeine synthesis] was
determined to be located here) and then is most probably transported
somewhere? I think the vacuole. I have made this conclusion due to literature
stating the presence of alkaloids in the vacuole.

The problem with the vacuole is that it contains large amounts of phenolics in
tea! And a hypothesis exists that phenolics and alkaloids co-aggregate in the
vacoule and if this is true I think it would make it almost impossible to
determine that caffeine is present in the vacoule.

This is quite a difficult project as there is a lot to consider but I am confident
something will work!

Any other ideas are welcome!

Thanks for the help!

Shane van Breda.
MSc Biochemistry.
University of Pretoria.
Department of Biochemistry.

[Rosemary.White]

Hi all,

Hmmm, since caffeine can be used to help stabilise phenolic compounds during
fixation, maybe phenolics, e.g. tannic acid, could be used to stabilise
caffeine?  Should give a dark deposit in the cells, but may also hide
epitopes recognised by the antibody.  At least it'd give an idea about the
caffeine-containing vacuoles or whatever they are.

cheers,
Rosemary

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

T 02 6246 5475
F 02 6246 5334
E [hidden email]



On 2/06/10 1:32 AM, "Martin Wessendorf" <[hidden email]> wrote:

> Dear Guy et al--
>
> Never having done a bit of work in plants, I have no idea of the
> considerations involved here.  However, based on the chemistry of
> caffeine--i.e., it's soluble in polar solvents and has no free primary
> amines or carboxyl groups--it seems as if it'd be unlikely to stay in
> place without fixation but hard to fix covalently.  The only way I know
> that might fix the stuff would be with McLean-Nakane fixation, where the
> ketones would get oxidized to aldehydes, but that would probably render
> it unrecognizable by the antibody.
>
> Is there any way that polarized microscopy could be used, since caffeine
> is birefringent?  I realize that starch is also birefringent, but is
> there likely to be much starch in tea leaves?
>
> Martin
>
> Guy Cox wrote:
>> I have immuno-labelled leaf epidermal peels.  I found that mild
>> paraformaldehyde fixation and cellulase treatment were necessary to get
>> penetration.  I can't answer for what that will do for caffeine, since I
>> don't know how easy it is to leach (though we do use boiling water to
>> make coffee or tea) but short of microinjection I can't see what would
>> do better.  Anything that would allow the antibody to enter would surely
>> allow caffeine to dissolve, it's just a question of how fast it
>> dissolves at room temperature.  Another point - leaf tissue after this
>> sort of treatment is not robust, so I found it was essential to fix the
>> epidermis to coverslips with poly-l-lysine.  Otherwise, even if they
>> looked OK, the intracellular structure was scrambled.
>>
>>                                            Guy
>>
>> Optical Imaging Techniques in Cell Biology
>> by Guy Cox    CRC Press / Taylor & Francis
>>      http://www.guycox.com/optical.htm
>> ______________________________________________
>> Associate Professor Guy Cox, MA, DPhil(Oxon)
>> Australian Centre for Microscopy & Microanalysis,
>> Madsen Building F09, University of Sydney, NSW 2006
>>
>> Phone [phone]     Fax [phone]
>>              Mobile [phone]
>> ______________________________________________
>>       http://www.guycox.net
>>  
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]]
>> On Behalf Of Shane van Breda
>> Sent: Monday, 31 May 2010 7:18 PM
>> To: [hidden email]
>> Subject: Immuno-labelling leaf material. Please help!
>>
>> I am currently trying to determine the tissue and intracellular
>> localisation of
>> caffeine in C.sinensis (tea) leaves. I have a polyclonal caffeine
>> antibody Ig
>> fraction that is directly labelled to FTC-ED.
>>
>> I am struggling to find literature of labelling leaves with
>> flourescently labelled
>> antibodies. Would it be possible to label fresh material without fixing
>> or
>> dehydration (I am worried these steps will dissolve or remove caffeine
>> from
>> the leaf).
>>
>> Alternatively I will be doing high pressure freezing and freeze
>> substitution (in a
>> solvent that caffeine does not dissovle in) or maybe freeze drying in 3
>> weeks
>> to embedd my samples. But how would I ensure labelling with my antibody?
>>
>> and will I still need to premeablise my cells?
>>
>> Another idea I have is to determine if caffeine has any type of
>> autoflourescence and then use this to aid in visualising it.
>>
>> Any ideas, references or protocols that could give me a starting point
>> will be
>> much appreciated!

[Guy Cox-2]

Well, except that tea contains a lot of phenolics already.  But then
again, they might stabilise the caffeine without any further
treatment????

                                          Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net
 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Rosemary White
Sent: Monday, 7 June 2010 4:55 PM
To: [hidden email]
Subject: Re: Immuno-labelling leaf material. Please help!

Hi all,

Hmmm, since caffeine can be used to help stabilise phenolic compounds
during
fixation, maybe phenolics, e.g. tannic acid, could be used to stabilise
caffeine?  Should give a dark deposit in the cells, but may also hide
epitopes recognised by the antibody.  At least it'd give an idea about
the
caffeine-containing vacuoles or whatever they are.

cheers,
Rosemary

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

T 02 6246 5475
F 02 6246 5334
E [hidden email]



On 2/06/10 1:32 AM, "Martin Wessendorf" <[hidden email]> wrote:

> Dear Guy et al--
>
> Never having done a bit of work in plants, I have no idea of the
> considerations involved here.  However, based on the chemistry of
> caffeine--i.e., it's soluble in polar solvents and has no free primary
> amines or carboxyl groups--it seems as if it'd be unlikely to stay in
> place without fixation but hard to fix covalently.  The only way I
know
> that might fix the stuff would be with McLean-Nakane fixation, where
the
> ketones would get oxidized to aldehydes, but that would probably
render
> it unrecognizable by the antibody.
>
> Is there any way that polarized microscopy could be used, since
caffeine
> is birefringent?  I realize that starch is also birefringent, but is
> there likely to be much starch in tea leaves?
>
> Martin
>
> Guy Cox wrote:
>> I have immuno-labelled leaf epidermal peels.  I found that mild
>> paraformaldehyde fixation and cellulase treatment were necessary to
get
>> penetration.  I can't answer for what that will do for caffeine,
since I
>> don't know how easy it is to leach (though we do use boiling water to
>> make coffee or tea) but short of microinjection I can't see what
would
>> do better.  Anything that would allow the antibody to enter would
surely
>> allow caffeine to dissolve, it's just a question of how fast it
>> dissolves at room temperature.  Another point - leaf tissue after
this
>> sort of treatment is not robust, so I found it was essential to fix
the

>> epidermis to coverslips with poly-l-lysine.  Otherwise, even if they
>> looked OK, the intracellular structure was scrambled.
>>
>>                                            Guy
>>
>> Optical Imaging Techniques in Cell Biology
>> by Guy Cox    CRC Press / Taylor & Francis
>>      http://www.guycox.com/optical.htm
>> ______________________________________________
>> Associate Professor Guy Cox, MA, DPhil(Oxon)
>> Australian Centre for Microscopy & Microanalysis,
>> Madsen Building F09, University of Sydney, NSW 2006
>>
>> Phone [phone]     Fax [phone]
>>              Mobile [phone]
>> ______________________________________________
>>       http://www.guycox.net
>>  
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List

[mailto:[hidden email]]

>> On Behalf Of Shane van Breda
>> Sent: Monday, 31 May 2010 7:18 PM
>> To: [hidden email]
>> Subject: Immuno-labelling leaf material. Please help!
>>
>> I am currently trying to determine the tissue and intracellular
>> localisation of
>> caffeine in C.sinensis (tea) leaves. I have a polyclonal caffeine
>> antibody Ig
>> fraction that is directly labelled to FTC-ED.
>>
>> I am struggling to find literature of labelling leaves with
>> flourescently labelled
>> antibodies. Would it be possible to label fresh material without

fixing
>> or
>> dehydration (I am worried these steps will dissolve or remove
caffeine
>> from
>> the leaf).
>>
>> Alternatively I will be doing high pressure freezing and freeze
>> substitution (in a
>> solvent that caffeine does not dissovle in) or maybe freeze drying in
3
>> weeks
>> to embedd my samples. But how would I ensure labelling with my
antibody?
>>
>> and will I still need to premeablise my cells?
>>
>> Another idea I have is to determine if caffeine has any type of
>> autoflourescence and then use this to aid in visualising it.
>>
>> Any ideas, references or protocols that could give me a starting
point
>> will be
>> much appreciated!

No virus found in this incoming message.
Checked by AVG - www.avg.com
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04:35:00

[Christian-103]

With the billion dollar coffee industry to interested in caffeine, is there anything similar in the literature?  A quick check of google scholar pops up a couple hundred possible articles.  Is there something different about what's out there and what you're trying to do?  If so it may help with protocol suggestions.

Christian



--- On Mon, 6/7/10, Guy Cox <[hidden email]> wrote:

From: Guy Cox <[hidden email]>
Subject: Re: Immuno-labelling leaf material. Please help!
To: [hidden email]
Date: Monday, June 7, 2010, 3:17 AM

Well, except that tea contains a lot of phenolics already.  But then
again, they might stabilise the caffeine without any further
treatment????

                                          Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
______________________________________________
      http://www.guycox.net


-----Original Message-----
From: Confocal Microscopy List [mailto:CONFOCALMICROSCOPY@...]
On Behalf Of Rosemary White
Sent: Monday, 7 June 2010 4:55 PM
To: CONFOCALMICROSCOPY@...
Subject: Re: Immuno-labelling leaf material. Please help!

Hi all,

Hmmm, since caffeine can be used to help stabilise phenolic compounds
during
fixation, maybe phenolics, e.g. tannic acid, could be used to stabilise
caffeine?  Should give a dark deposit in the cells, but may also hide
epitopes recognised by the antibody.  At least it'd give an idea about
the
caffeine-containing vacuoles or whatever they are.

cheers,
Rosemary

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

T 02 6246 5475
F 02 6246 5334
E rosemary.white@...



On 2/06/10 1:32 AM, "Martin Wessendorf" <martinw@...> wrote:

> Dear Guy et al--
>
> Never having done a bit of work in plants, I have no idea of the
> considerations involved here.  However, based on the chemistry of
> caffeine--i.e., it's soluble in polar solvents and has no free primary
> amines or carboxyl groups--it seems as if it'd be unlikely to stay in
> place without fixation but hard to fix covalently.  The only way I
know
> that might fix the stuff would be with McLean-Nakane fixation, where
the
> ketones would get oxidized to aldehydes, but that would probably
render
> it unrecognizable by the antibody.
>
> Is there any way that polarized microscopy could be used, since
caffeine
> is birefringent?  I realize that starch is also birefringent, but is
> there likely to be much starch in tea leaves?
>
> Martin
>
> Guy Cox wrote:
>> I have immuno-labelled leaf epidermal peels.  I found that mild
>> paraformaldehyde fixation and cellulase treatment were necessary to
get
>> penetration.  I can't answer for what that will do for caffeine,
since I
>> don't know how easy it is to leach (though we do use boiling water to
>> make coffee or tea) but short of microinjection I can't see what
would
>> do better.  Anything that would allow the antibody to enter would
surely
>> allow caffeine to dissolve, it's just a question of how fast it
>> dissolves at room temperature.  Another point - leaf tissue after
this
>> sort of treatment is not robust, so I found it was essential to fix
the

>> epidermis to coverslips with poly-l-lysine.  Otherwise, even if they
>> looked OK, the intracellular structure was scrambled.
>>
>>                                            Guy
>>
>> Optical Imaging Techniques in Cell Biology
>> by Guy Cox    CRC Press / Taylor & Francis
>>      http://www.guycox.com/optical.htm
>> ______________________________________________
>> Associate Professor Guy Cox, MA, DPhil(Oxon)
>> Australian Centre for Microscopy & Microanalysis,
>> Madsen Building F09, University of Sydney, NSW 2006
>>
>> Phone [phone]     Fax [phone]
>>              Mobile [phone]
>> ______________________________________________
>>       http://www.guycox.net
>> 
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List

[mailto:CONFOCALMICROSCOPY@...]

>> On Behalf Of Shane van Breda
>> Sent: Monday, 31 May 2010 7:18 PM
>> To: CONFOCALMICROSCOPY@...
>> Subject: Immuno-labelling leaf material. Please help!
>>
>> I am currently trying to determine the tissue and intracellular
>> localisation of
>> caffeine in C.sinensis (tea) leaves. I have a polyclonal caffeine
>> antibody Ig
>> fraction that is directly labelled to FTC-ED.
>>
>> I am struggling to find literature of labelling leaves with
>> flourescently labelled
>> antibodies. Would it be possible to label fresh material without

fixing
>> or
>> dehydration (I am worried these steps will dissolve or remove
caffeine
>> from
>> the leaf).
>>
>> Alternatively I will be doing high pressure freezing and freeze
>> substitution (in a
>> solvent that caffeine does not dissovle in) or maybe freeze drying in
3
>> weeks
>> to embedd my samples. But how would I ensure labelling with my
antibody?
>>
>> and will I still need to premeablise my cells?
>>
>> Another idea I have is to determine if caffeine has any type of
>> autoflourescence and then use this to aid in visualising it.
>>
>> Any ideas, references or protocols that could give me a starting
point
>> will be
>> much appreciated!

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