Re: Safranin-O staining on EDTA decalcified rat bone

From:Gayle Callis <>

EDTA is used by biochemists to extract proteoglycans from cartilage, and
that is what has happened to the proteoglycans in your articular cartilage
during your decalcification. There is nothing that will recover what is
removed.  It is better to use the acid decalcifier, and some publications
recommend buffered formic acid or and acidic buffer as you did.  You need
to endpoint test used EDTA solution, hopefully you have xray, fastest, most
sensitive as chemical method for EDTA is longer.  You would have to acidify
the EDTA to force release of calcium, then do a traditional chemical
endpoint test. 

If you left the bones in 'worry free' EDTA method too long, more
proteoglycans are removed, and endpoint testing should tell you when Ca is
gone without overexposure to EDTA. I have seen articular cartilage
decalcified with EDTA with some loss of staining, although prefer formic
acid/buffered for my own protocol. A good control to gear the eye for what
you want to see, even with acidic decalcification, is a piece of
UNDECALCIFIED articular cartilage stained with SAFO. There may be some
proteoglycan loss with acid decalcifiers also per   references I have on
the subject, but not as much as with EDTA. 

The original SAFO method was done by Rosenberg on undecalcifed cartilage,
frozen sections, probably to avoid any proteoglycan loss.   

At 01:33 PM 5/17/01 -0400, you wrote:
>I have formalin fixed paraffin embedded rat bone that has been decalcified
>in 14% EDTA.  I am looking at articular cartilage on the stifle joint.  When
>we did our routine Safranin-O, the EDTA decalcified bone did not pick up the
>Safranin-O.  Sections decalcified with formic acid and stained in the same
>batch worked beautifully.  Does the EDTA bone need some sort of
>pretreatment?  I have tried 1% magnesium chloride for 2 hours but this
>didn't help.
>Kim Kowsz
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Gayle Callis
Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
Bozeman MT 59717-3610

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