[Histonet] Cryosectioning of retina/choroid/sclera?

[Srikant Tripathy]

Hi,

Thanks for your reply.

No, I do not want to fix the layers in fixative like formalin or
Paraformaldehyde as these are diluted solutions and may dissolve the soluble
ion I am trying to estimate. Secondly to fix these tissues I need to take a
thin section and this may disturb the ion concentration results.

Is it possible for cryosectioning the fragile retina after snap freezing of
fresh tissue, with out cryoprotectant?

Many Thanks again,
Srikant.

-----Original Message-----
From: Gayle Callis [mailto:gcallis <@t> montana.edu] 
Sent: Tuesday, 12 December 2006 2:42 AM
To: Srikant Tripathy
Subject: Re: [Histonet] Cryosectioning of retina/choroid/sclera?

Are you fixing the layers first with either formalin or 
paraformaldehyde?   That is when you need cryoprotection, after total 
fixtaion in 30% sucrose overnight or longer at 4C.  Then you can snap 
freeze.  If you are freezing the tissues in fresh state, you do NOT need 
cryoprotection.  These fixatives are often buffered with sodium and 
potassium phosphate salts, so beware, you may NOT want to add more elements 
to the system.

At 06:36 PM 12/5/2006, you wrote:
>Hi,
>
>I am Srikant a PhD student in Queensland University of
>Technology,Brisbane,Australia.In a part of my study I am trying to estimate
>the element concentration (Na,K,Cl) at various layers of eye
>(retina,choroid,sclera) by EDX analysis and confocal  microscope.
>
>I am a Pharmacist and don't have very good knowledge of histological
>techniques.
>
>
>
>  I am using cryosectioning of eye layers and for a fragile layer like
retina
>I need to cryoprotect before sectioning. I am using snap freezing
>technology.
>
>
>
>My doubts are:
>
>1.      As I can't use water soluble cryoprotectant as this will leach all
>soluble ion. I wonder how people can keep retina soaked in Ames's solution
>and gets fluorescence for chloride ions using dyes like MEQ.
>2.      Can any one please guide me how I will proceed for cryoprotecting
>and sectioning the retina/choroid/sclera with out adverse effects on
>elemental concentration?
>3.      Can I use PEG as a coating material prior to cryosectioning for
>elemental analysis before EDX and confocal microscopy?
>
>
>
>Thanks.
>
>Srikant
>
>
>
>
>
>Srikant Tripathy | PhD Student | Institute of Health and Biomedical
>Innovation
>
>Queensland University of Technology | Corner of Musk Avenue and Blamey
>Street, Kelvin Grove QLD 4059 Australia
>
>t: +61 (0) 7  3138 6157 | f: +61 (0) 7 3138 6030 | m: 0423586276 | e:
>s.tripathy <@t> qut.edu.au <BLOCKED::mailto:s.tripathy <@t> qut.edu.au>   |  w:
><BLOCKED::http://www.ihbi.qut.edu.au/> www.ihbi.qut.edu.au
>
>
>
>CRICOS No. 00213J
>
>
>
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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)