>	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


Kelly,

	From the sound of it I don't think you are doing anything wrong
with this stain.  I worked at the U of Minnesota Orthopedic Lab for a few
years and this was my favorite stain.  Most often I would decalcify with
10% formic acid/10% formaldehyde.  I had some fairly large dog bones to
deal with.  We did find though that it only took a relatively small drop in
the concentration of Pg's to result in a total loss of staining of Safranin
O.  This was fine with us at the time, since we were looking for just such
a loss due to various outside effects.  With a small piece, such as mouse,
it would be very easy to reduce the Pg's to the point that they wouldn't
stain.
	If getting the cartilage to stain up is important to you, you could
always try Toluidine Blue O or Alcian Blue.  The other thing that you might
try is to add cetylpyridinium chloride (CPC)  0.5% to your fixative.  I
used to used this as a matter of course, but to tell the truth I don't have
any real proof that it helped me.
	You also didn't mention the decal times.  For mouse bones 10%
formic should get the job done overnight.  Make sure those guys are well
fixed before the decal though.
	That S.O/F.G is a pretty stain.  I especially liked the effect in
the zone of calcified cartilage where you could see the tide mark effect.
You could easily see the difference between the newly forming lamelar bone
and the more amorphous "calcified cartilage". I used to take some pictures
using this stain and the polarizer that people just loved. The good old
days.

Randy Carpenter




Parker Hughes Cancer Center
rcarpenter@ih.org
randallcarpenter@qwest.net (home)