Fwd: ALP for histonet

From:John Baker

Hi all,  This message is from a graduate student in our laboratory. 
Thanks in advance for any help you can give her.  My best,  John

>X-Sender: ncaldwel@n.imap.itd.umich.edu
>Date: Thu, 4 Oct 2001 16:24:23 -0400
>To: bakerj@umich.edu
>From: Nancy Caldwell 
>Subject: ALP for histonet
>I am wondering if you can give me some advice/suggestions to help me work
>out the Alkaline Phosphatase staining protocol I have been working on. We
>have been attempting to get ALP staining on paraffin sections of
>decalcified trabecular bone. I have seen a few examples in the literature,
>as well as in some histology books (Bancroft and Cook, 1994) which we
>caught on to from histonet listings.
>Specifically, we have tried the BCIP-NBT (5-Bromo-4-chloro-3-indolyl
>phoshpate - Nitro blue tetrazolium chloride) method from p. 300 of Bancroft
>and Cook (Manual of histological techniques and their diagnostic
>application, 1994) [a similar method is in Histochemistry in Pathology,
>Filipe and Lake, 1990]. This method involves and incubation medium
>containing BCIP-NBT, veronal acetate buffer, and magnesium chloride.
>We have tried the method described in JOR 16:636-642 by Tay et. al. which
>utilizes an Alk Phos Buffer (Tris, MgCl2, NaCl, and Tween 20- all at
>specific pH) and then incubation in bromcresol purple.
>We have also tried using the method we have previously worked with on GMA
>embedded specimens with napthol AS-BI as the substrate and Fast Red Violet
>as the coupler in a Sigma diagnostics ALP kit.
>We can get all of these to work on cryo-sections, but as of yet haven't had
>any success on decalcified paraffin-embedded sections (and the paraffin
>sections are obviously much quicker and easier to obtain, as well as
>getting more reliable sections).  We have been able to get TRAP staining
>(which looks quite nice) on the paraffin sections using a Sigma diagnostics
>kit (Napthol AS-BI as the substrate and Fast Garnet GBC Base solution as
>the coupler). We have tried a variety of decalcification agents, including
>FASC, AFS (with pHs ranging from 4 to 5), and EDTA (pHs ranging from 4.6 to
>6.5, and sometimes using overnight incubation in a 1% MgCl2 solution to try
>to reactivate the enzyme).
>Do you have any advice/suggestions/experience that might be helpful?
>Thanks!   Nancy Caldwell

John A. Baker
The University of Michigan
Orthopaedic Research Laboratories
Histology Unit
400 North Ingalls, G160
Ann Arbor, MI 48109-0486
Main lab office phone:734-763-9674
Histology office:734-936-1635

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