[Histonet] Re: Massons Trichrome on decalcified bone

Jack Ratliff ratliffjack <@t> hotmail.com
Thu Apr 5 09:06:32 CDT 2012

Hello Sara!

Might I quickly ask why you decalcify instead of processing and embedding undecalcified using methyl methacrylate resin/plastic? I routinely process these type of specimens into MMA and cut either 5 micron sections without metal present or thick/ground sections polished to 30-40 microns if a metallic device is present.

These same specimens that have been processed and embedded into plastic and cut at 5 microns can be deplasticized prior to staining with H&E, Goldner's trichrome, VonKossa-MacNeal's tetrachrome, Safranin O-Fast Green, Sirius Red-Fast Green, etc. If these specimens contain a large metal implant, the sections are stained undeplasticized with Sanderson's Rapid Bone Stain & Van Gieson picrofuchsin.

Of course of you don't have these capabilities or equipment to process these specimens into resin, decalcification is your only option, but I would personally use 5% or 10% Formic acid instead of the lengthy EDTA process. I would also use methyl salicylate to replace the xylenes steps so that you can avoid making the bone too brittle and difficult to cut.

Feel free to message me back if you need further explanation or if you would even like to discuss privately by phone. I would also be happy to forward you stained images of these stains, previously listed and from this same specimen type you are working with, that has been resin embedded. Also, I have trained people all over the world to process undecalcified bone (any size) into resin/plastic and cut at 5 microns if this is an option for you!

Best Regards,


Jack Ratliff
Hard Tissue Histologist
Chairman, Hard Tissue Committee - National Society for Histotechnology 

On Apr 5, 2012, at 9:20 AM, Sara Landschoot <sllandsc <@t> gmail.com> wrote:

> Hi Gayle,
> Thanks so much for your response on Histonet. I have been getting several
> different answers that I have been trying to test.
> I can give you more details on how we decal our bone here. We deal with
> entire sheep or primate spines which are grossed while frozen into slabs.
> The slabs are about 5-6mm thick. We take an initial xray to get a starting
> point then we decal the slabs in EDTA (after formalin fixation). Xrays are
> taken throughout the decal process to check for the endpoint.
> As for post fixing in Bouins, I was using the water bath method (60
> degrees) for about an hour.
> If you don't mind sending me the AFIP methods so I can try then out on my
> sections I would greatly appreciate it. My email is sllandsc <@t> gmail.com.
> Again thank you for your help and I look forward to hearing from you.
> Sara Landschoot
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