Re: Alkaline Phosphatase in paraffin?
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.
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: firstname.lastname@example.org [SMTP:email@example.com]
> > 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
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