[Histonet] question about decalicification end-point checks

Rene J Buesa rjbuesa <@t> yahoo.com
Mon Oct 8 11:49:00 CDT 2012

What you describe (NBF fixation → band saw slicing → decal with formic acid → end point with 5% ammonium oxalate) seems a good protocol BUT you have to pay special attention to:
1- Did you fix the femurs "in toto"? If you did that, fixation is extremely difficult. You first should select a "general area" of interest and isolate that area with the band saw in a way that the NBF can penetrate the bone via the medullar area, otherwise fixation will be incomplete and start a "chain of irreversible problems".
2- assuming that the fixation of the whole bone was good, how thick the band saw slices to be decalcified were? They should be 5 mm thick at the most.
3- if the 2 previous issues were not completed that way you may have a (+) test for "complete decalcification" that may not reflect the reality, but lets assume the bone slices were decalcified correctly then
4- infiltration has to be extended at twice the time of your standard protocol and with a paraffin wax of at least 63ºC of melting point or ideally higher. You may not have that paraffin wax in your lab, but this is a special subject that requires special reagents if you want to arrive to a successful final section.
5- the rest will be microtomy related preferably using steel "permanent" knives, no disposable blades, with the adequate cutting angle and, ideally, with a sliding microtome 
As you can see there are many variables and even under the best circumstances, bone microtomy is very difficult.
I never recommend  "after standard protocol solutions" like "treating the exposed cutting surface" with acid solutions. These are desperate attempts to compensate for procedural mistakes that will never render ideal sections.
René J.

From: "Thurby, Christina" <christina.thurby <@t> bms.com>
To: "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu> 
Sent: Monday, October 8, 2012 11:35 AM
Subject: [Histonet] question about decalicification end-point checks

I am looking for help with decalcification end-point checks.  We use a 5% Formic acid decal (homemade - 1 L 88% Formic acid, 1 L 37% Formaldehyde and 8 L of distilled water).  We do an endpoint check using 5% ammonium oxylate.  Recently, we decaled canine femurs for a total of 13 days and endpoint check was performed on days 12 and 13.  The femurs were fixed in 10% NBF and then sectioned with a band saw prior to decalcification.  The decal endpoint tested out on both days 12 and 13.  Our problem is at microtomy, the center of the bones do not appear to be completely decaled and we can not get a good section.  We have tried surface decaling the blocks with no luck.  Any thoughts?  Is there a commercially available decal endpoint check that others have used for acid decals?

I have just finished reading Mr. Skinner & Ms. Schray's article "Decalicification of Mouse Bone/Soft Tissue Composit Specimens" from mikro-graf publishing, Volume 41, Issue 01  - this article does give some good information about paraffin embedding (which may be part of our issue on these larger bone specimens).  Perhaps we may look into the 50/50 embedding medium at a higher temp for large animal bone specimens like this in the future which seem to yield good results in Mr. Skinner's laboratory.

Robert or Carrie - if one of you are available could you give me a call or e-mail to discuss?  Thanks so much!!  Any feedback will be greatly appreciated!

Kristie Thurby
Bristol Myers Squibb

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