Hi Barry,
	Could you use isopropanol instead of acetone? It can be used for
dehydrating as well as clearing. Thanks Jenny

-----Original Message-----
From: Barry Rittman [mailto:brittman@mail.db.uth.tmc.edu]
Sent: Monday, February 18, 2002 3:09 PM
To: histology
Subject: Re: Alkaline Phosphatase in paraffin?


One point to mention is that while this method works it was recommended for
tissue high in alkaline phosphatase i.e. kidney etc. If using
acetone/paraffin
method there is a loss of between 50 and 60% of the alkaline phosphatase.
Tissues fixed in acetone do not cut very well but the rationale for using
acetone is that it is miscible with paraffin wax.
Barry

"Smith, Allen" wrote:

> George Gomori used to fix tissues in acetone or ethanol, embed in 56
degree
> paraffin, section, and stain for alkaline phosphatase.  Gomori, G. (1946)
> Am.J.Clin.Path.16:177.  Gomori, G. (1951) MICROSCOPIC HISTOCHEMISTRY.
> Fredricsson, B. (1956) Acta Anatomica 26:246.  See also volume 1 of
> Pearse's HISTOCHEMISTRY, THEORETICAL AND APPLIED.
>
> Allen A. Smith, Ph.D.
> Barry University
> School of Graduate Medical Sciences
>     Podiatric Medicine and Surgery
> Miami Shores, Florida  33161-6695
>
> -----Original Message-----
> From: rueggp [mailto:rueggp@earthlink.net]
> Sent: Saturday, February 16, 2002 11:00 AM
> To: Dan Beniker
> Cc: 'hborgeri@wfubmc.edu'; 'Histonet'
> Subject: Re: Alkaline Phosphatase in paraffin?
>
> Dan,
> I don't think the alk.p enzyme method will work on decalcified paraffin
> sections.  I have always done it either on frozen sections or samples
fixed
> in
> cold methanol and embedded in GMA resin without decalcification.  For
> paraffin
> sections the IHC methods may be your only option.
> Patsy Ruegg
>
> Dan Beniker wrote:
>
> > Thanks for the quick response, Hermina! I've tried an antibody for AP
> before
> > and had mixed results.... Maybe the antibody needs revisiting! What I
> > neglected to mention is that I'm trying for an enzymatic stain....Hoping
> to
> > stain using BCIP/NBT or fast red, etc. I've been able to get good
results
> > using BCIP/NBT on cryos, but have tried several fixation/decal
techniques
> > (Formalin vs EtOH fixation and EDTA vs Formic Acid Decal) on paraffin
and
> > haven't had anything work. I read somewhere that the process of paraffin
> > embedding (the heat involved?) inactivates the enzyme, but have also
heard
> > from others who have apparently had good results in paraffin....
> >
> > > -----Original Message-----
> > > From: hborgeri@wfubmc.edu [SMTP:hborgeri@wfubmc.edu]
> > > Sent: Friday, February 15, 2002 4:46 PM
> > > To:   Dan Beniker
> > > Cc:   'Histonet'
> > > Subject:      Re: Alkaline Phosphatase in paraffin?
> > >
> > > Hi Dan,
> > >
> > > Please see my publication in the Journal of Histotechnology, Volume
15,
> > > No.2,
> > > June 1992.  The antibody I used for the demonstration of alkaline
> > > phosphatase
> > > was a gift from Dr. Larry Fisher(BLK ALP [LF-47]) and worked very
well.
> > > EDTA
> > > demineralization chelates calcium ions, causing the inactivation of
ALP.
> > > By
> > > using magnesium chloride, I was able to reactivate the enzyme, which
was
> > > then
> > > detected using streptavidin-conjugated beta galactosidase.
> > >
> > > Hermina
> > >
> > > Hermina
> > >
> > > Dan Beniker wrote:
> > >
> > > > I'm interested in protocols for successful staining of bone alkaline
> > > > phosphatase in fixed, decalcified, paraffin embedded samples. I've
> heard
> > > > that it can be done, but have not had any success. Currently, the
only
> > > way I
> > > > can get alk phos to work is in zinc formalin fixed, EDTA decalcified
> > > > cryosections. If anyone has a protocol or some pointers for working
> this
> > > > magic, I'd really be interested!
> > > >
> > > > Thanks!
> > > >
> > > > Dan Beniker
> > > > LifeCell Corp
> > > > One Millennium Way
> > > > Branchburg, NJ 08876
> > > > (908) 947-1175 (voice)
> > > > (908) 947-1085 (fax)
> > >
> > > --
> > > Hermina Borgerink, BA, HTL, HT(ASCP)IHQ
> > > Department of Pathology
> > > Wake Forest University School of Medicine
> > > Winston-Salem, N.C. 27157
> > > PH (336) 716-1538
> > > Fax (336) 716-1515
> > >
> > >