Safranin O on articular cartilage, loss of staining

From:Gayle Callis

First a question:

Did you run a piece of articular cartilage that was NEVER decalcified?
This is a must in order to know how your decalcifiers affect the
proteoglycans you are trying to stain. Basically, a normal, untreated
control tissue. I am trying to recall, someone can fill in the blanks here,
but the proteoglycans in growth plate and articular cartilage consist of
some of the same and different types.  You also see differences in staining
of these two cartilage types with H and E stains, in particular, Ehrlichs
hematoxylin and also with toludine blue staining.    

EDTA WILL extract proteoglycans (biochemists have been using this method
for years in order to do chemical analysis on proteoglycans removed from
the tissue).  EDTA extraction obviously changes tinctorial levels of
staining with Saf O Fast green - something we observed in a 1998
publication after testing several acid and chelating decalcifying agents.
This is also in the literature ie the effects of decalcifying agents on
proteoglycans.  Acid decalcifiers can also affect proteoglycans and the
acidic buffers ie formic acid/sodium formate or sodium citrate have been
used with good success. 

It is interesting to note that the SAF O/ staining method was origianally
done on articular cartilage using undecalcified, fresh frozen sections.


 At 09:16 AM 12/11/01 -0500, you wrote:
>	I've been working on a safranin o/fast green stain (for cartilage)
>and only getting very faint safranin o staining in the articular cartilage,
>though the growth plate in these bones stains a very bright red.  I'd
>appreciate any advice anyone might have on how to improve this staining...
>	As a brief overview, this is what I'm doing right now:
>	Mouse knee/elbows/paws were decalcified in EDTA, formic acid or
>nitric acid and paraffin embedded.  They were then deparaffinized,
>rehydrated and stained with Weigert's hematoxylin, counterstained with fast
>green and decolorized in 1% acetic acid briefly.  Following this, the
>sections were stained for 10-20 minutes in a 0.1% commercial safranin o
>solution (in 0.1% acetic acid), dehydrated, cleared and coverslipped.
>	Thanks in advance.
>Kelly A. Beckwith
>Research Associate, Histology
>Cardion Pharmaceuticals, Inc
Gayle Callis
Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367
406 994-4303 (FAX)

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