I only need to adjust the pH to 6.5 to 7.0 or so to save on sodium
hydroxide.  Believe me, at hot boiling temperature, EDTA will dissolve very
quickly, about 10-15 minutes.  And just add sodium hydroxide in to keep them
in solution.  The reason why I have MgCl2 in is to keep my proteoglycan
staining in Safranin-O.  It worked well for me compared to without it.  We
used to have to use formic acid to decal to get good staining of
proteoglycan in the cartilage, but not anymore.

I use this decal solution to decal quite big tissues.  Femoral heads of
immature to mature pigs, with thickness around 0.5 -1 cm, and area of around
2-3cm (the section will occupy 3/4 area on a slide), most of them are bone.
I change the solution once in the morning, once in the evening before
leaving, at RT.  It will usually take about 5-7 days to decal.  I've never
tried on biopsy punches, but my guess is several hours to 1 day??? I
normally check for complete decal with X-ray instead of using ammonium
hydroxide and ammonium oxalate, because X-ray is so much more sensitive and
accurate.  It's expensive, but after a while, I would have some idea when
the tissues are completely decalcified.  So far we have very good results
with IHC, TUNEL...  Hope this help.

> -----Original Message-----
> From:	Abizar Lakdawalla [SMTP:abizarl@innogenex.com]
> Sent:	Wednesday, August 23, 2000 1:00 PM
> To:	Morken, Tim
> Cc:	'Histonet'
> Subject:	Re: IP's with Decal
> 
> EDTA at that concentration (12-14%) may only go into solution after the pH
> is
> adjusted to 8 (adjusted with sodium hydroxide). It is not very common to
> add
> divalent ions to the EDTA reagent, as the main purpose of EDTA is to
> chelate and
> remove divalent ions (like Ca and Mg) from the bone/cartilage.
> Abizar
> www.innogenex.com
> 1-877-IGX-INFO
> 
> "Morken, Tim" wrote:
> 
> > So how long does it take to decal with this mixture?
> >
> > -----Original Message-----
> > From: Su, Phy-Huynh [mailto:psu@shctampa.usf.edu]
> > Sent: Tuesday, August 22, 2000 5:42 PM
> > To: 'Histonet'
> > Subject: RE: IP's with Decal
> >
> > I am using EDTA buffered to decal, too.  I made it myself to have a
> higher
> > concentration of EDTA to decal faster.  Typically I'll have 12.% to 14%
> EDTA
> > in the solution.  Boil your water in a microwave and pour EDTA powder
> in.
> > That will dissolve EDTA very quickly.  Cool it down, then buffer it, and
> add
> > other stuff.  You can have your 4% paraformaldehyde, or 10% NBF in the
> > solution (if you fix your tissues using those) to fix and decal at the
> same
> > time.  I also have some MgCl2 in my decal solution.  (Only add MgCl2
> after
> > the solution was buffered.)  This seems to help to keep my Safranin-O
> > staining for proteoglycan good.
> >
> > Other people decal at 37 degree, or using microwave.  This seems to
> speed up
> > decal time.  But I have lost some antigenicity this way, so I prefer to
> do
> > it at RT or at 4 degree with stirring.
> >
> > If you want to try my recipe, e-mail me.
> > Good luck
> > Su
> >
> > > -----Original Message-----
> > > From: Abizar Lakdawalla [SMTP:abizarl@innogenex.com]
> > > Sent: Tuesday, August 22, 2000 1:07 PM
> > > To:   Jennifer Englin
> > > Cc:   histonet@pathology.swmed.edu
> > > Subject:      Re: IP's with Decal
> > >
> > > EDTA (versene) based decal solutions work best for most immuno's.
> > > Downside: it's expensive and takes much more longer to decal.
> > > abizar
> > > www.innogenex.com
> > > 1-877IGX-INFO
> > >
> > > Jennifer Englin wrote:
> > >
> > > > Has anyone found a decal that works well with immuno's?
> > > > We are currently using Stephens Scientific Decal- formic
> > > acid/formaldahyde/mathanol, and we are having trouble with our Kappa
> and
> > > Lambda.
> > > > Any suggestions?
> > > >
> > > > Jennifer,
> > > > Willmar MN
> > >
>