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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 <[log in to unmask]> wrote:

> *****
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> 
> 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: [log in to unmask]
> To: [log in to unmask]
> Subject: Re: Embedding Arabidopsis Thaliana for confocal
> 
> ***** 
> To join, leave or search the confocal microscopy listserv, go to: 
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> 
> 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: 
> 
> 
>> ***** 
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>> 
>> 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 
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