SPAM-LOW: RE: [Histonet] Canine bone sectioning trouble

[Patsy Ruegg]

Oh my, I agree with Liz, your samples are way too thick and your process is
way too short.  Even for 3-4mm thickness bone I fix for 24-72 hours and then
decal in 5% formic acid usually overnight on a platform shaker.  I assume
RDO decals a lot faster but not that fast, I do not use RDO because I think
the formic acid which may be slower better preserves the antigens for IHC.

Patsy

Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net 
www.ihcrg.org


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Liz Chlipala
Sent: Wednesday, June 30, 2010 5:36 PM
To: Schneider,Lynda S; histonet <@t> lists.utsouthwestern.edu
Subject: SPAM-LOW: RE: [Histonet] Canine bone sectioning trouble

Overall your fixation, decalcification and processing cycle is too
short.  The sample size is really big, why do you need it so thick?  I
would first of all let the samples fix longer 24 to 48 hours or I would
trim them so that they are about 3-5 mm in thickness. Either way I like
fixing bone samples for 24 to 48 hours.  I'm not that accustomed to
using RDO decal it is hydrochloric acid based and is quicker but even
bone that size would take a while to decal.  We normally use a formic
acid based decal and a sample size that large my take up to 2 weeks to
decal.  Also to process a sample that size I would be at 4 to 6 hours a
station.  We frequently process porcine and goat knees in 2 x 3 blocks
the samples are quite thick about a cm in thickness and we will process
them at 4 to 6 hours a station.

My recommendation would be to decal the larger cube of bone for a period
of time until you can section through it.  I would then cut a 3-4 mm
thick piece off the cube and process it at 1 hour per station.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com
 
 
Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
Schneider,Lynda S
Sent: Tuesday, June 29, 2010 10:18 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Canine bone sectioning trouble

Hello out there...

We are having trouble sectioning canine bone.  The samples are bone
marrow cubes approximately  2cm thick and 1in wide.  They were fixed for
15 hours in 10% NBF and decaled for 30 - 45 mins in RDO rapid
decalcifier.  They were faced in and then surface decaled again for
about 30 mins.  When sectioned, much of the marrow was missing and the
bone was torn and shredded.  We thought that maybe the samples had not
been fixed sufficiently so refixed overnight in 10% NBF again.  The
samples were then reprocessed as they had been originally.  The
processing schedule used was: 70% EtOH 30mins, 80% EtOH 30mins, 95% EtOH
x 2, 30mins each, 100% EtOH x 2, 30 mins each, xylene x 2, 45 mins each,
paraffin x 4, 40 mins each. Again they were faced in and decaled for
another 30 mins or so.  This time as soon as the sections are placed on
the water bath (38 degrees) they explode and/or come apart so severely
sections can almost not be picked up.  If sections are even obtainable
they are of horrible quality.  Does anyone have any suggestions? Thank
you so much in advance!

Lynda
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