Re: Mounting non fixed tissue ? **tech support response**

[Roy Edward-2]

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Dear Jean-Pierre,

You may wish to consider CyGEL.  A recent relevant example using this
technology is the imaging of fresh excised mosquito gut tissue (Wilson,
R., et al., PLoS neglected tropical diseases 4.9 (2010): e816).

CyGEL is a thermo-reversible mountant, designed specifically for live
cells, tissues and intact organisms (Danio embryos, C elegans, Drosophila
embryos, parasites).  Unusually, it is a liquid when cold and a gel when
warmed above 22 degC, and infinitely thermo-reversible allowing
repositioning and recovery after imaging.  It is water-based, giving it
excellent optical properties with a RI of 1.37, being clear and having no
appreciable auto-fluorescence in the visible range.

I¹ve used this for a lot of different cells and organisms.

You can use this with or without a coverslip.  The usual technique is to
overlay CyGEL onto the tissue section, placing the coverslip on top and
then chilling to allow the gel to liquefy and spread under the coverslip.
Finally, allow the slide to warm and the CyGEL to set once more. The
coverslip can be sealed if required.

Alternatively, you can glue two coverslips down onto a slide to allow
bridging of a third coverslip to top the CyGEL if your material is
delicate and you want to avoid crushing it (e.g. a rapidly dividing
Drosophila embryo as demonstrated by Judy Callaghan, Monash Univ).

Thirdly, you could create a ³well² on a slide using a silicone
grease-smeared silicone o-ring.  Bed this onto a slide placing your
tissue/object inside and overlay with CyGEL.  A further emulation is to do
this in a coverslip-bottomed petri-dish and surround the o-ring with LMP
agarose as a moisture buffer for extended imaging (e.g. overnight) thereby
avoiding desiccation of the CyGEL and sample held within.  This has proved
particularly useful for Danio imaging.

You can dope CyGEL with a nuclear counterstain (e.g. DRAQ5), viability dye
(e.g. PI, DRAQ7), organelle stain (e.g. Lysotracker) or anaesthetic such
as MS-222.  

You may wish to refer to:
Price, H.P., et al.
Validation of a new method for immobilising kinetoplastid parasites for
live cell imaging.
Molecular and Biochemical Parasitology 169.1 (2010): 66-69

or full information is available at www.biostatus.com
<http://www.biostatus.com>

I assume you are in Japan - BioStatus products can be sourced in Japan
from Cosmo Bio Ltd. or otherwise worldwide direct from BioStatus or
selected distribution partners.

Kind regards,
Roy

Roy Edward
BioStatus Limited
56a Charnwood Road, Shepshed, Leicestershire LE12 9NP
T [phone] | F [phone]
www.biostatus.com

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>-----Original Message-----

>From: Jean-Pierre CLAMME [mailto:[hidden email]]
>Sent: venerd=EC 8 novembre 2013 20:11
>Subject: Mounting non fixed tissue ?
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=3Dconfocalmicroscopy
>*****
>
>Hi,
>
>I was wondering if anyone knows about the possibility of mounting thin
>tiss=
>ue sections without fixing the tissue. What would be a protocol for such
>sa=
>mples ?
>All the protocol I've found involve fixing.
>
>Thank you.
>
>JP