| From: | Tony Henwood |
Dear Yichao,
I would transfer the
renal biopsies in a cell culture fluid (eg Hanks), place them in an esky with
ice if you have to travel some distance.
I recommend you RAPIDLY freeze
them in OCT using Liquid Nitrogen or some similar FAST freezing method (I believe the main problem here is the slow freezing offered by the
cryostat).
If you are staining them H&E, try immediate methanol fixation of the frozen sections.
If doing immunofluorescence, then air dry
for 20-30 minutes prior to staining.
Other comments
below.
Hope this
helps,
Tony Henwood JP, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory
Manager
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: (02) 9845 3306
Fax: (02) 9845
3318
http://www.histosearch.com/homepages/TonyHenwood/default.html
http://us.geocities.com/tonyhenwoodau/index.html
-----Original
Message-----
From: yichao wu [mailto:yichaowu@hotmail.com]
Sent:
Tuesday, 3 June 2003 12:54
To: histonet@pathology.swmed.edu
Subject: How
could I get better frozen slides?
In our lab,frozen sections from
kidney specimens always look poor.I want to
know how other labs
do.
Our procedures are as below,
Firstly we get specimens from clinic
bedside.
Take them back to our lab in a 4C box.
Immerse them in
OCT.
Put directly in -29C cyrostat and wait for them to be
frozen.
Sectioning.
Use slides in room temperature to stick to those sectioned tissue. And
frozen sections are prepared.
I wonder,
1)
Should we use lower temperature when transfering the specimens? Through
lower
temperature is very inconvenient. NO
2) Should we incubate the specimens in
sucrose buffer before immerse them in
OCT? How to do it in details?
Not necessary
3) Should we change
a cyrostat? (Ours is from Bright and have used it for
about 10 yrs) If you rapidly freeze the tissue and the cryostat is
working well, there is no need.
Could you kindly give
some suggestions? We just wish to have frozen sections
of better quality.Such
as could identify basement membrane and glomeruli in
the setions.Now all we
could see is a mass of cells. What thickness are you
cutting? Try thinner
Thank you very much!
Yichao
WU,Ph.D candidate
Research Insititute of Nephrology,Jinling
Hospital
Medical School,Nanjing University
Nanjing 210002
PR
China
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