[Histonet] Re: Decalcification and processing of large bone

[Prior, Andrew]

I work with bone samples trimmed to approx 35mmx35mmx4mm. Our general procedure is:

Fixation:
untrimmed samples stored in 10% NBF @4°C until trimming. Slices placed in fresh 10% NBF for at least 12h.

Decalcification
Benchtop - 10% formic acid for 10 days (change soln every 3 days), rinse for minimum 1-2 hours. Then 10% EDTA solution for 2-3 weeks (change weekly). The time in formic acid is limited at 10 days to prevent excess damage to tissue/loss of nuclear staining. The EDTA is a chelating agent and is slower at decalcifying tissue but does little damage.
Microwave processor - 3 days in 10% formic acid @35°C, rinse as above, then 3 days in 10% EDTA @35°C.
For both methods I recommend placing a layer of empty cassettes at the bottom of you container to allow solution to circulate under samples. After the EDTA step, it is VITAL that samples are rinsed for at least 3 hours to remove any remainng salts. These precipitate during processing and make sectioning very difficult. You may need to leave samples longer in EDTA depending on their size. There are various methods in text books for determining decalcification end point. We have a MicroCT scanner so just do a quick xray scan to check our samples.

Processing.
We have a  Sakura Tissue Tek 5. Samples processed for 5 hours each  through 50%, 70%, 90% and 3x100% Ethanol/IMS. Then 4 hours x3 changes for chloroform, then 2 hours x4 changes of wax.

Sectioning
Make sure block are well chilled. Some samples may need soaking in water to get good sections. We trim in samples then place face down on a melting ice block whilst trimming in the other samples. This means our samples soak and chill at the same time. If you get small areas of mineral left, you can perform surface decal with 10% fromic acid.

Hope this points you in the right direction. If you can, try and go to this years NSH convention. Lots of very helpful people there, especially the Hard Tissue Committee. Best of luck.

Andrew Prior
Histologist
Smith &Nephew Research Centre
York Science Park
UK
 
Andrew.Prior <@t> smith-nephew.com
 
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Message: 21
Date: Wed, 24 Feb 2010 17:34:06 -0800 (PST)
From: Victor Wong <vhlwong <@t> yahoo.com>
Subject: [Histonet] Decalcification and processing of large bone
To: Histonet <@t> lists.utsouthwestern.edu
Message-ID: <347173.34928.qm <@t> web52706.mail.re2.yahoo.com>
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Dear all,
 
I am going to decal and process PIG femur and lumbar vertebrates.  I only have experience on soft tissues.  Anyone can suggest any protocols in decalcification and processing?  Many thanks in advance.
 
Cheers,
Victor

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