[Histonet] Help with frozen sections

[Alan Bright]

Dear Deirdre,

 

You do not state the temperature of the tissue you are trying to section
in the cryostat. I think that raising the temperature would eliminate
the cracking.

 

Best Regards

Alan Bright

Bright Instrument Co.Ltd.
St Margaret's Way
Huntingdon
Cambridgeshire
PE29 6EU
England

Tel No:+44 (0)1480 454528
Fax No:+44 (0)1480 456031
Email: abright <@t> brightinstruments.com
Web Site: www.brightinstruments.com

 

-----Original Message-----
From: Deirdre Brophy [mailto:deirdre_brophy <@t> hotmail.com] 
Sent: 28 October 2003 12:43
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Help with frozen sections

 

Hello,
Does anyone have any advice on getting good frozen sections from tissue
that has been stored in formaldehyde for prolonged periods? 

I am currently cutting fish gonads on a cryostat. This could potentially
save a lot of time and expense compared to wax histology. My samples
have been stored in 4% buffered formaldehyde for several months. Due to
the nature of my sampling, this is difficult to avoid. I have been
cryoprotecing the tissue with a mixture of DMSO and sucrose, and then
freezing in a bath of hexane cooled with dry ice. I am taking 6-10um
sections which are then stained with H&E and viewed with a light
microscope. For the female gonads, this is working quite well, the
morphology is good and cryprotection seems to keep freeze damage to a
minimum. However, for the testes the results are not so good. Sections
are cracked and morphology is really poor. When the same tissue is cut
using wax histology the sections are fine. Rinsing the tissue well in
PBS to remove excess formaldehyde before sectionning on the cryostat
helps, as does cryprotecting for longer periods (overnight) but cracks
are s till visible. Could prolonged storage in formaldehyde render the
tissue less permeable to the cryoprotectant perhaps? Any
suggestions/remedies would be much appreciated as I am running out of
things to try!

Best wishes,

Deirdre

 

_______________________

Deirdre Brophy

Dept of Life Sciences

GMIT

Dublin rd

Galway

Ireland




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