vibratome of fresh eye tissue?

[Mary Ellen Pease]

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I am interested in sectioning human optic nerve (unfixed) with a vibratome
(without freezing or embedding). Is this feasible? I am having difficulty finding
recent information regarding vibratoming fresh tissues online. It seems most lightly
fix and or embed their tissues in gelatin before sectioning.

thanks!
Mary Ellen Pease
Wilmer Eye Institute
Johns Hopkins School of Medicine

[Martin Wessendorf-2]

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Dear Dr. Pease--

On 7/15/2014 3:02 PM, Mary Ellen Pease wrote:
> I am interested in sectioning human optic nerve (unfixed) with a vibratome
> (without freezing or embedding). Is this feasible? I am having difficulty finding
> recent information regarding vibratoming fresh tissues online. It seems most lightly
> fix and or embed their tissues in gelatin before sectioning.
Check with a slice electrophysiologist--they do this sort of thing all
the time (albeit not in human tissue).  There should be a bunch of them
in the Neuroscience department at Hopkins.

Basically, you would need to glue the optic nerve to a chuck with
superglue.  You would put a small block of agar immediately behind the
nerve to steady it during cutting, so that it doesn't get bent over from
the force of the blade.

Safety first: since this is human tissue, use a splash shield over the
vibratome bath, and of course wear gloves.

Good luck!

Martin Wessendorf

--
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]

[Tobias Baskin]

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Mary Ellen,
                     For what it is worth, we section plant organs on a
vibratome, fresh, without fixation, routinely. We glue them on the stub
with superglue (!) and then after that sets, add a drop of buffer which
immerses the sample, and slice away. Very useful method.

         Good vibes!
                                 Tobias

On 7/15/14, 4:02 PM, Mary Ellen Pease 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.
> *****
>
> I am interested in sectioning human optic nerve (unfixed) with a vibratome
> (without freezing or embedding). Is this feasible? I am having difficulty finding
> recent information regarding vibratoming fresh tissues online. It seems most lightly
> fix and or embed their tissues in gelatin before sectioning.
>
> thanks!
> Mary Ellen Pease
> Wilmer Eye Institute
> Johns Hopkins School of Medicine
>

--
       __    ___   ^    ___   ___  Tobias I. Baskin
      /  \  /     / \  /      \     Professor
     /   / /     /   \ \       \     Biology Department
    / __/ /__   /___  \ \       \__   611 N. Pleasant St.
   /     /     /       \ \       \     University of Massachusetts
  /     /     /         \ \       \     Amherst, MA, 01003 USA
/     /___  /           \ \___/   \_____    413 - 545 - 1533
   www.bio.umass.edu/biology/baskin

[Glen MacDonald-2]

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HI Mary Ellen,
We vibratome fresh brain routinely.   Different investigators have different small modifications to the same basic techniques.  Once the brain is blocked, where the unneeded portion is sliced off to provide both preferred orientation and a flat mounting surface, excess liquid is removed and it is attached to the mount with superglue.  Some glues work better than others.  Aaron Alpha is good,  lately we’ve been using Vigor CS-480.  A block of agarose is glued behind the tissue as a backstop.  For embryonic brains, a sapphire blade was much better than a razor.  Usually, the brain is attached to a plastic petri dish glued to the mounting stub.  The dish is filled with appropriate media,  and the bath chamber of the vibratome is filled with crushed ice.  Slow cutting speed (rate of advance) and higher amplitude is usually best for us, but finding the optimal combination for each tissue, species and developemental stage will be by trial and error. Cutting angle is also very important.  

Good luck.

Glen MacDonald
        Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
        Cellular Morphology Core
Center on Human Development and Disability
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]







On Jul 16, 2014, at 7:08 AM, Tobias Baskin <[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.
> *****
>
> Mary Ellen,
>                    For what it is worth, we section plant organs on a vibratome, fresh, without fixation, routinely. We glue them on the stub with superglue (!) and then after that sets, add a drop of buffer which immerses the sample, and slice away. Very useful method.
>
>        Good vibes!
>                                Tobias
>
> On 7/15/14, 4:02 PM, Mary Ellen Pease 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.
>> *****
>>
>> I am interested in sectioning human optic nerve (unfixed) with a vibratome
>> (without freezing or embedding). Is this feasible? I am having difficulty finding
>> recent information regarding vibratoming fresh tissues online. It seems most lightly
>> fix and or embed their tissues in gelatin before sectioning.
>>
>> thanks!
>> Mary Ellen Pease
>> Wilmer Eye Institute
>> Johns Hopkins School of Medicine
>>
>
> --
>      __    ___   ^    ___   ___  Tobias I. Baskin
>     /  \  /     / \  /      \     Professor
>    /   / /     /   \ \       \     Biology Department
>   / __/ /__   /___  \ \       \__   611 N. Pleasant St.
>  /     /     /       \ \       \     University of Massachusetts
> /     /     /         \ \       \     Amherst, MA, 01003 USA
> /     /___  /           \ \___/   \_____    413 - 545 - 1533
>  www.bio.umass.edu/biology/baskin

[Mary Ellen Pease]

*****
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*****

thanks so much for your feedback!
I think I can try this pretty easily.
take care!
Mary Ellen


On Tue, Jul 15, 2014 at 5:09 PM, Martin Wessendorf <[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 Dr. Pease--
>
>
> On 7/15/2014 3:02 PM, Mary Ellen Pease wrote:
>
>> I am interested in sectioning human optic nerve (unfixed) with a vibratome
>> (without freezing or embedding). Is this feasible? I am having difficulty
>> finding
>> recent information regarding vibratoming fresh tissues online. It seems
>> most lightly
>> fix and or embed their tissues in gelatin before sectioning.
>>
> Check with a slice electrophysiologist--they do this sort of thing all the
> time (albeit not in human tissue).  There should be a bunch of them in the
> Neuroscience department at Hopkins.
>
> Basically, you would need to glue the optic nerve to a chuck with
> superglue.  You would put a small block of agar immediately behind the
> nerve to steady it during cutting, so that it doesn't get bent over from
> the force of the blade.
>
> Safety first: since this is human tissue, use a splash shield over the
> vibratome bath, and of course wear gloves.
>
> Good luck!
>
> Martin Wessendorf
>
> --
> 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]
>

--
*Mary Ellen*
-------------
Mary Ellen Pease
410-292-2551 (cell)
[hidden email]

[Mary Ellen Pease]

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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*****

thanks Tobias - good "vibes" to you as well!


On Wed, Jul 16, 2014 at 10:08 AM, Tobias Baskin <[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.
> *****
>
> Mary Ellen,
>                     For what it is worth, we section plant organs on a
> vibratome, fresh, without fixation, routinely. We glue them on the stub
> with superglue (!) and then after that sets, add a drop of buffer which
> immerses the sample, and slice away. Very useful method.
>
>         Good vibes!
>                                 Tobias
>
>
> On 7/15/14, 4:02 PM, Mary Ellen Pease 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.
>> *****
>>
>> I am interested in sectioning human optic nerve (unfixed) with a vibratome
>> (without freezing or embedding). Is this feasible? I am having difficulty
>> finding
>> recent information regarding vibratoming fresh tissues online. It seems
>> most lightly
>> fix and or embed their tissues in gelatin before sectioning.
>>
>> thanks!
>> Mary Ellen Pease
>> Wilmer Eye Institute
>> Johns Hopkins School of Medicine
>>
>>
> --
>       __    ___   ^    ___   ___  Tobias I. Baskin
>      /  \  /     / \  /      \     Professor
>     /   / /     /   \ \       \     Biology Department
>    / __/ /__   /___  \ \       \__   611 N. Pleasant St.
>   /     /     /       \ \       \     University of Massachusetts
>  /     /     /         \ \       \     Amherst, MA, 01003 USA
> /     /___  /           \ \___/   \_____    413 - 545 - 1533
>   www.bio.umass.edu/biology/baskin
>

--
*Mary Ellen*
-------------
Mary Ellen Pease
410-292-2551 (cell)
[hidden email]