Protoplast confocal

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Filiz Gurel Filiz Gurel
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Protoplast confocal

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Dear All;
I look for a procedure simply to prepare slides for analyzing plant protoplasts under confocal microscopy? Specifically working on Yfp.  I appreciate any help.
Benjamin Smith Benjamin Smith
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Re: Protoplast confocal

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Since we were using an inverted confocal, we just used a coverslip bottom
dish such as the following: https://www.mattek.com/store/p35g-1-5-10-c-case/

For an upright confocal, you could use the same dish with a coverslip on
top, or if you are running out of working distance, put a ring of
petrolatum on a glass slide, put the protoplasts in the ring with water,
and then gently press down the coverslip.

-Ben Smith

On Mon, Nov 6, 2017 at 8:30 AM, Filiz Gurel <[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.
> *****
>
> Dear All;
> I look for a procedure simply to prepare slides for analyzing plant
> protoplasts under confocal microscopy? Specifically working on Yfp.  I
> appreciate any help.
>



--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
Tel  (510) 642-9712
Fax (510) 643-6791
e-mail: [hidden email]
http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>
Rosemary.White Rosemary.White
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Re: Protoplast confocal

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Hi Filiz,

We regularly look at protoplasts on an upright confocal; just put a small drop of solution plus protoplasts on a slide then add a coverslip with a spacer(s) of some sort to prevent the weight of the coverslip from crushing the protoplasts.

The spacer between slide and coverslip can be
- a ring of wax from a pap pen
- four dots of nail polish, one placed on each corner of a small square coverslip and allowed to dry completely before use
- four dots of vaseline/petroleum jelly placed on the corners of a coverslip - this is good because you can gently push the coverslip down until it's the right height above your protoplasts
- slivers of coverslip of about the diameter of your protoplasts, placed at each end under the coverslip on the protoplasts - have used this quite a bit also.

The trick is to have enough liquid to keep the protoplasts happy but not so much that they slide around during imaging.

If this is hard to get right, they can also be embedded in low melting point agarose in your osmoticum plus buffer of choice. This ancient paper gives an example https://link.springer.com/content/pdf/10.1007/BF01323276.pdf

cheers,
Rosemary

On 7/11/17, 5:37 am, "Confocal Microscopy List on behalf of Benjamin E Smith" <[hidden email] on behalf of [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.
    *****
   
    Since we were using an inverted confocal, we just used a coverslip bottom
    dish such as the following: https://www.mattek.com/store/p35g-1-5-10-c-case/
   
    For an upright confocal, you could use the same dish with a coverslip on
    top, or if you are running out of working distance, put a ring of
    petrolatum on a glass slide, put the protoplasts in the ring with water,
    and then gently press down the coverslip.
   
    -Ben Smith
   
    On Mon, Nov 6, 2017 at 8:30 AM, Filiz Gurel <[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.
    > *****
    >
    > Dear All;
    > I look for a procedure simply to prepare slides for analyzing plant
    > protoplasts under confocal microscopy? Specifically working on Yfp.  I
    > appreciate any help.
    >
   
   
   
    --
    Benjamin E. Smith, Ph. D.
    Imaging Specialist, Vision Science
    University of California, Berkeley
    195 Life Sciences Addition
    Berkeley, CA  94720-3200
    Tel  (510) 642-9712
    Fax (510) 643-6791
    e-mail: [hidden email]
    http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>
   

Filiz Gurel Filiz Gurel
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Re: Protoplast confocal

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Thank you very much !