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Embedding Arabidopsis Thaliana for confocal

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annaoru

Embedding Arabidopsis Thaliana for confocal

Hi!
I´m a student doing my exam-project where we are using Arabidopsis Thaliana to express a HIV protein (p24) in order to creat an edible vaccine.
Previously I have fixated and embedded (paraffin wax) the tissues to vizualise it in Light microscope (via IHC). Now I need to do the same procedure but try to analyze the tissue using confocal microscope. For that I need to know if I can use the same fixation solution for confocal as I did for Light microscope which was: egual volume of 0.2M Sodiumphosphate buffer pH 7.2 and Paraformaldehyde (8%) solution?
And also what kind of embedding should I use for the confocal?
Since I have a lot of material left from my previous work I´m hoping to be able to use it as it is for the confocal.
Is this possible?

Thank you for replying!

/Anna

George McNamara

Re: Embedding Arabidopsis Thaliana for confocal

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Hi Anna,

Only way to know is to do the experiment.

I have a suggestion for transitioning from IHC to fluorescence confocal
microscopy: assuming you are using HRP with DAB + H2O2, you can
substitute DAB with a fluorescent tyramide (available from
Invitrogen/Molecular Probes,
http://probes.invitrogen.com/media/pis/mp20911.pdf ) or PerkinElmer Life
Sciences. You can ignore the other components in the kit (store those
as directed in the kit). Pick a fluorophore in a wavelength range that
has low autofluorescence for your specimen (one of your current fixed
specimens - ex. a negative control). I strongly encourage the use of
serial sections, with alternating sections detected with DAB vs tyramide.

Sincerely,

George
p.s. Your current DAB precipitate IHC specimens might be suitable for
reflection (backscattered) confocal microscopy. DAB ppt's scatter
strongly in the blue (405 nm, 440 nm, 458 nm laser lines). You may need
to look up (or ask your vendor) your confocal microscope settings to get
a good backscatter signal. You can also visualize DAB on the confocal by
transmitted light (not a confocal mode). If you routinely counterstain
with hematoxylin, you can even acquire an RGB transmitted light image,
i.e. 458 nm for blue, 514 nm for green, 633 nm for red.

On 1/24/2011 5:27 AM, annaoru wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi!
> I´m a student doing my exam-project where we are using Arabidopsis Thaliana
> to express a HIV protein (p24) in order to creat an edible vaccine.
> Previously I have fixated and embedded (paraffin wax) the tissues to
> vizualise it in Light microscope (via IHC). Now I need to do the same
> procedure but try to analyze the tissue using confocal microscope. For that
> I need to know if I can use the same fixation solution for confocal as I did
> for Light microscope which was: egual volume of 0.2M Sodiumphosphate buffer
> pH 7.2 and Paraformaldehyde (8%) solution?
> And also what kind of embedding should I use for the confocal?
> Since I have a lot of material left from my previous work I´m hoping to be
> able to use it as it is for the confocal.
> Is this possible?
>
> Thank you for replying!
>
> /Anna
>

--

George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

Lloyd Donaldson

Re: Embedding Arabidopsis Thaliana for confocal

Anna

Arabidopsis influorescence stems or short shoots can be embedded in LR White without problem provided you cut the stems in half so they are not completely surrounded in cuticle. I haven't tried leaves - might need vacuum to assist with infiltration. Have you tried confocal on your wax embedded material ?

Regards

Dr Lloyd Donaldson

Senior Scientist, Project Leader - Microscopy/Wood Identification
Scion - Next Generation Biomaterials
Private Bag 3020, Rotorua
New Zealand 3010

Ph: 64 7 343 5581

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of George McNamara
Sent: Tuesday, 25 January 2011 12:36 a.m.
To: [hidden email]
Subject: Re: Embedding Arabidopsis Thaliana for confocal

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Anna,

Only way to know is to do the experiment.

I have a suggestion for transitioning from IHC to fluorescence confocal
microscopy: assuming you are using HRP with DAB + H2O2, you can
substitute DAB with a fluorescent tyramide (available from
Invitrogen/Molecular Probes,
http://probes.invitrogen.com/media/pis/mp20911.pdf ) or PerkinElmer Life
Sciences. You can ignore the other components in the kit (store those
as directed in the kit). Pick a fluorophore in a wavelength range that
has low autofluorescence for your specimen (one of your current fixed
specimens - ex. a negative control). I strongly encourage the use of
serial sections, with alternating sections detected with DAB vs tyramide.

Sincerely,

George
p.s. Your current DAB precipitate IHC specimens might be suitable for
reflection (backscattered) confocal microscopy. DAB ppt's scatter
strongly in the blue (405 nm, 440 nm, 458 nm laser lines). You may need
to look up (or ask your vendor) your confocal microscope settings to get
a good backscatter signal. You can also visualize DAB on the confocal by
transmitted light (not a confocal mode). If you routinely counterstain
with hematoxylin, you can even acquire an RGB transmitted light image,
i.e. 458 nm for blue, 514 nm for green, 633 nm for red.

On 1/24/2011 5:27 AM, annaoru wrote:

--

George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

Disclaimer: This e-mail and any attachments may contain information which is confidential or subject to copyright. If you receive this e-mail in error, please delete it.
Scion does not accept responsibility for anything in this e-mail which is not provided in the course of Scion’s usual business or for any computer virus, data corruption, interference or delay arising from this e-mail.

annaoru

Re: Embedding Arabidopsis Thaliana for confocal

Hi!
No I haven´t tried yet but I think I might try just to see the results.
I haven´t succeeded to find any information regarding this and the possibilities around using the same embedding and fixation technique as I did prior to Light microscopy but I hope that it will work or that I at least can use the same fixation so that the embedded tissue can be melt and re-embedded in resin instead. What do you think about that?

Thank you for helping!

Regards!
/Anna

Ryan Geil

Re: Embedding Arabidopsis Thaliana for confocal

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

It is possible to reverse paraffin embedded material back down through your infiltration series and then re-embed in LR White but it is very time consuming and can be hard on the tissue as extra handling and processing are required. It is much more advisable to start from fresh material and go through a proper LR White infiltration. That being said, I would recommend both paraformaldehyde and glutaraldehyde for fixation for a good mix of fast penetration and good cross-linking. I would also use much smaller tissue sections for LR White processing than I would for paraffin embedding. In my experience, vacuum infiltration is absolutely necessary when working with any leaf tissue. I did a lot of embedding work in a past life and I very likely have a protocol kicking around somewhere. If you are interested in having a look, let me know and I will dig it up.
Cheers,
Ryan Geil

On 2011-01-24, at 4:16 PM, annaoru wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi!
> No I haven´t tried yet but I think I might try just to see the results.
> I haven´t succeeded to find any information regarding this and the
> possibilities around using the same embedding and fixation technique as I
> did prior to Light microscopy but I hope that it will work or that I at
> least can use the same fixation so that the embedded tissue can be melt and
> re-embedded in resin instead. What do you think about that?
>
> Thank you for helping!
>
> Regards!
> /Anna
> --
> View this message in context: http://confocal-microscopy-list.588098.n2.nabble.com/Embedding-Arabidopsis-Thaliana-for-confocal-tp5954747p5956682.html
> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.

George McNamara

Re: Embedding Arabidopsis Thaliana for confocal

In reply to this post by annaoru

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Anna,

If you are happy with the morphology of your IHC sections, my
fluorescent tyramide suggestion will cost you some money to purchase the
kit, but enable you to avoid other changes.

best wishes,

George

On 1/24/2011 4:16 PM, annaoru wrote:

--

George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

annaoru

RE: Embedding Arabidopsis Thaliana for confocal

In reply to this post by Ryan Geil

Thank you so much Ryan!
I would be very excitet if you had a protocol somewhere, but only if it wouldn´t be too much work for you finding it!
I will talk to my supervisor and see what they think based on your advise.

thank you again!

regards!
/Anna

Date: Mon, 24 Jan 2011 13:47:12 -0800
From: [hidden email]
To: [hidden email]
Subject: Re: Embedding Arabidopsis Thaliana for confocal

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

It is possible to reverse paraffin embedded material back down through your infiltration series and then re-embed in LR White but it is very time consuming and can be hard on the tissue as extra handling and processing are required. It is much more advisable to start from fresh material and go through a proper LR White infiltration. That being said, I would recommend both paraformaldehyde and glutaraldehyde for fixation for a good mix of fast penetration and good cross-linking. I would also use much smaller tissue sections for LR White processing than I would for paraffin embedding. In my experience, vacuum infiltration is absolutely necessary when working with any leaf tissue. I did a lot of embedding work in a past life and I very likely have a protocol kicking around somewhere. If you are interested in having a look, let me know and I will dig it up.
Cheers,
Ryan Geil

On 2011-01-24, at 4:16 PM, annaoru wrote:

If you reply to this email, your message will be added to the discussion below:

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annaoru

RE: Embedding Arabidopsis Thaliana for confocal

In reply to this post by George McNamara

Okej George!
How interesting!
How is it working and if I would use that technique can a re-use the tissue I already have fixated and embedded or do I need fresh tissue?

Thank you for helping!

Regards
/Anna

Date: Mon, 24 Jan 2011 17:16:57 -0800
From: [hidden email]
To: [hidden email]
Subject: Re: Embedding Arabidopsis Thaliana for confocal

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Anna,

If you are happy with the morphology of your IHC sections, my
fluorescent tyramide suggestion will cost you some money to purchase the
kit, but enable you to avoid other changes.

best wishes,

George

On 1/24/2011 4:16 PM, annaoru wrote:

--

George McNamara, PhD
Analytical Imaging Core Facility
University of Miami

If you reply to this email, your message will be added to the discussion below:

http://confocal-microscopy-list.588098.n2.nabble.com/Embedding-Arabidopsis-Thaliana-for-confocal-tp5954747p5957211.html

To unsubscribe from Embedding Arabidopsis Thaliana for confocal, click here.

Dobeck, Justine

Re: Embedding Arabidopsis Thaliana for confocal

In reply to this post by annaoru

Have successfully done confocal using 6 micron paraffin sections of decalcified mouse molar and incisor teeth which had been fixed in 10% buffered formalin. Had used some of the sections to do IHC using DAB as substrate with Vector ABC kit. The only difference in protocols was that quenched in 2% sodium borohydride in PBS for 1.5 hours changing the solution at 45 minutes and then rinsed 5 times in PBS before proceeding with blocking step. Sodium borohydride quenches much of the endogenous autofluorescence from aldehydes. Antibodies were to e and n cadherins and used Alexafluors 488 and 568. You might want to increase concentration of primary and secondary from what used for light microscopy.

Justine
Justine M. Dobeck
Biostructure Core Facility
Rm. 5123
The Forsyth Institute
245 First Street
Cambridge, MA 02142
(617)892-8316
Email: [hidden email]

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of annaoru
Sent: Monday, January 24, 2011 5:27 AM
To: [hidden email]
Subject: Embedding Arabidopsis Thaliana for confocal

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi!
I´m a student doing my exam-project where we are using Arabidopsis Thaliana
to express a HIV protein (p24) in order to creat an edible vaccine.
Previously I have fixated and embedded (paraffin wax) the tissues to
vizualise it in Light microscope (via IHC). Now I need to do the same
procedure but try to analyze the tissue using confocal microscope. For that
I need to know if I can use the same fixation solution for confocal as I did
for Light microscope which was: egual volume of 0.2M Sodiumphosphate buffer
pH 7.2 and Paraformaldehyde (8%) solution?
And also what kind of embedding should I use for the confocal?
Since I have a lot of material left from my previous work I´m hoping to be
able to use it as it is for the confocal.
Is this possible?

Thank you for replying!

/Anna
--
View this message in context: http://confocal-microscopy-list.588098.n2.nabble.com/Embedding-Arabidopsis-Thaliana-for-confocal-tp5954747p5954747.html
Sent from the Confocal Microscopy List mailing list archive at Nabble.com.

Ryan Geil

Re: Embedding Arabidopsis Thaliana for confocal

In reply to this post by annaoru

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

I have had good success with the following embedding protocol for leaf tissue. It is a really good idea to do several changes in LR White for several days to ensure good infiltration and prevent the tissue from separating from the cuticle in your sections.
You may want to avoid the glut as it is a very strong cross-linker. The leaf tissue for embedding should be harvested in a drop of fixative on dental wax so that the fixative gets in immediately. Remember, good micro- technique requires time and patience. There are a lot of steps afterwards to get to imaging so you want to make sure you start off properly.

Fixation:

For Structure: 2% Paraformaldehyde + 2% Glutaraldehyde in 50mM PIPES pH 7.0

For Labeling: 3% Paraformaldehyde in 50mM PIPES pH 7.0

Fix under vacuum for 1-2h (then leave at 4°C overnight)

Dehydration:

Rinse in 50mM PIPES pH 7.0 3 x 10min

20% EtOH 1h

50% EtOH 1h

70% EtOH 1h

95% EtOH 1h

Infiltration:

2:1 (95% EtOH:LR White) 1h

1:1 1h

1:2 1h

100% LR White 1h

100% LR White 3d

100% LR White 3d

Polymerization: 1.5-2h at 55-60°C

Cheers
Ryan Geil

On 2011-01-25, at 12:42 AM, annaoru <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Thank you so much Ryan!
> I would be very excitet if you had a protocol somewhere, but only if it wouldn´t be too much work for you finding it!
> I will talk to my supervisor and see what they think based on your advise.
>
> thank you again!
>
> regards!
> /Anna
>
>
>
> Date: Mon, 24 Jan 2011 13:47:12 -0800
> From: [hidden email]
> To: [hidden email]
> Subject: Re: Embedding Arabidopsis Thaliana for confocal
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> It is possible to reverse paraffin embedded material back down through your infiltration series and then re-embed in LR White but it is very time consuming and can be hard on the tissue as extra handling and processing are required. It is much more advisable to start from fresh material and go through a proper LR White infiltration. That being said, I would recommend both paraformaldehyde and glutaraldehyde for fixation for a good mix of fast penetration and good cross-linking. I would also use much smaller tissue sections for LR White processing than I would for paraffin embedding. In my experience, vacuum infiltration is absolutely necessary when working with any leaf tissue. I did a lot of embedding work in a past life and I very likely have a protocol kicking around somewhere. If you are interested in having a look, let me know and I will dig it up.
> Cheers,
> Ryan Geil
>
> On 2011-01-24, at 4:16 PM, annaoru wrote:
>
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi!
>> No I haven´t tried yet but I think I might try just to see the results.
>> I haven´t succeeded to find any information regarding this and the
>> possibilities around using the same embedding and fixation technique as I
>> did prior to Light microscopy but I hope that it will work or that I at
>> least can use the same fixation so that the embedded tissue can be melt and
>> re-embedded in resin instead. What do you think about that?
>>
>> Thank you for helping!
>>
>> Regards!
>> /Anna
>> --
>> View this message in context: http://confocal-microscopy-list.588098.n2.nabble.com/Embedding-Arabidopsis-Thaliana-for-confocal-tp5954747p5956682.html
>> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.
>
>
>
>
>
>
> If you reply to this email, your message will be added to the discussion below:http://confocal-microscopy-list.588098.n2.nabble.com/Embedding-Arabidopsis-Thaliana-for-confocal-tp5954747p5956769.html
> To unsubscribe from Embedding Arabidopsis Thaliana for confocal, click here.
> --
> View this message in context: http://confocal-microscopy-list.588098.n2.nabble.com/Embedding-Arabidopsis-Thaliana-for-confocal-tp5954747p5957661.html
> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.