[Histonet] Paraffin processing native sheep ACL

[Andrew Prior]

Hi Liz,



I inherited the following protocol for ACL samples. It works quite well, but times probably could be reduced - the optimisation is on my to-do list.

70% Alcohol - 1 hour

90% alcohol - 1 hour

100% Alcohol -2 hours

100% alcohol - 3 hours

100% alcohol - 4 hours

Xylene               - 1.5 hours

Xylene               - 1.5 hours

Xylene               - 3 hours

Wax                   - 3 hours

Wax                   - 3 hours

Wax                   - 4 hours

I cut the sections at 8um so they hold together better. Takes a while for all the wrinkles to disappear when floating out on water-bath so be patient

Hope this helps.



Andrew


Andrew Prior
Histologist
Tissue Regenix Group
E-mail: a.prior <@t> tissueregenix.com<mailto:a.prior <@t> tissueregenix.com>
Website:  www.tissueregenix.com<http://www.tissueregenix.com/>



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Message: 3

Date: Wed, 12 Jun 2013 16:59:52 -0400

From: Elizabeth Ronan <lizronan <@t> umich.edu<mailto:lizronan <@t> umich.edu>>

Subject: [Histonet] Paraffin processing native sheep ACL

To: histonet <@t> lists.utsouthwestern.edu<mailto:histonet <@t> lists.utsouthwestern.edu>

Message-ID: <51B8E148.3020102 <@t> umich.edu<mailto:51B8E148.3020102 <@t> umich.edu>>

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Hello,



I need to paraffin process native sheep anterior cruciate ligament (ACL) that has been fixed in 10% neutral buffered formalin for 7 days. I was wondering if anyone with more expertise on this subject could guide me with the best lengths of times in the alcohols, xylenes, and paraffin to fix ACL. I tried a 12 hour program but the sections crumbled in the middle and it appeared that the paraffin had not fully perfused the ligament.



I have access to the following program, and can alter the lengths of the steps for as long as desired:



Program:

70%

80%

95%

95%

100%

100%

Xylene

Xylene

Paraffin

Paraffin

Paraffin



Any advice is much appreciated.

Thanks for your time,

Liz



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