[Histonet] Paraffin processing native sheep ACL

[Jack Ratliff]

You might also consider using methyl salicylate instead of xylenes. Thanks to the help of Bob Skinner, I have achieved very nice results with native tendon. 
Generally speaking these MS steps will take a little longer, but you can monitor the progress very easily by watching for complete transparency of the tendon. You can then even develop a somewhat standardized protocol if you plan to process this type of tissue in the future. You even have a lot more flexibility with MS than xylenes as prolonged use in xylenes can make the tissue more hardened and brittle.
Lastly, it is not generally recommended to put MS on the tissue processor, so I process to the final 100% EtOH, perform the MS exchanges by hand, transfer the tissues into a manual wax step to get rid of as much MS as possible and then finish with three (3) automated wax steps on the tissue processor.
For my wax infiltration I use a 50:50 blend of TissuePrep from Fisher Scientific and EM400 from Leica. I then embed in 100% EM400.
Best Regards,
Jack


Jack L RatliffOwner/Histologist, Ratliff Histology Consultants, LLCChairman, Hard Tissue Committee - National Society for Histotechnology



> From: a.prior <@t> tissueregenix.com
> To: histonet <@t> lists.utsouthwestern.edu
> Date: Fri, 14 Jun 2013 07:45:47 +0000
> Subject: Re: [Histonet] Paraffin processing native sheep ACL
> CC: 
> 
> Hi Liz,
> 
> 
> 
> I inherited the following protocol for ACL samples. It works quite well, but times probably could be reduced - the optimization 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/>
> 
> 
> 
> ------------------------------
> 
> 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>>
> 
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
> 
> 
> 
> 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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