[Histonet] Decal question

[Elizabeth Chlipala]

Jackie

We cut a lot of bone sections here, we experimented quite a bit with the
infiltration and paraffin embedding, and found that the harder the
infiltration paraffin the better the sections would stay on the slides,
no flipping of the articular cartilage.  We also found that the harder
paraffins made it difficult to section and obtain ribbons, so we had to
make some changes to the infiltration media to find one that we were
able to cut sections (get ribbons) and the sections would not flip
during staining.  We actually found a combination of surgipaths formula
R (1/3) and richard allens paraffin 9 (2/3) worked the best with
embedding in Richard allens paraffin 9.  We rough trim our sections and
let them sit on water and ice for about an hour or so.  Then we section
them and lay them flat on a hot plate, just like Patsy said, but we do
not let the paraffin melt.  They sit on the hot plate at about 36
degrees overnight, we do not place them in an oven to melt the paraffin,
but transfer directly to staining racks and stain.  We use plus slides
and we rarely get any flipping.  

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)
Premier Histology Laboratory, LLC
P.O. Box 18592 
Boulder, Colorado 80308
Office: (303) 735-5001
Fax: (303) 735-3540
lizchlipala <@t> premierhistology.com
www.premierhistology.com
 
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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
Jackie.O'Connor <@t> abbott.com
Sent: Wednesday, April 14, 2004 9:17 AM
To: Histonet (E-mail); histonet-bounces <@t> lists.utsouthwestern.edu
Subject: [Histonet] Decal question

I'm working on a trial with intra-tibial injections of tumors into mice.

Whole (skin and muscle trimmed) legs were fixed for about a month in 10%

NBF.  I used Surgipath's Decal I to decalcify (took about 6 hours - end 
point determined by cutting through femur with scalpel).  Paraffin 
sections cut nicely - I used a 48C waterbath to ensure no wrinkles,
picked 
up on positively charged slides.  I let them air dry overnight, then 60C

for an hour prior to H+E staining.  I'm not happy.  The tumors and 
cortical bone are lifting and falling off the slides.  Any suggestions? 
I've been reading old posts about EDTA decalcification - since hindsight

is 20/20 - would that have been a better choice??  Boo hoo.

Jackie O'
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