RE: [Histonet] TRAP Staining

From:"Hermina Borgerink"

Hi Nena,
This method works very well on both plastic and paraffin sections.

Tartrate-Resistant Acid Phosphatase (TRAP) histochemistry

For Paraffin:
Deparaffinize sections through xylenes and alcohol and bring to running deionized water for 5 minutes.  
For Plastic:
Deplasticize through three 20-minute changes of 2-methoxyethyl acetate, two 5-minute changes of acetone followed by running deionized water for 5 minutes.
Make up acetate buffer in the volume needed for the number of slides to be stained.
Distilled water                      for     50.0 ml     for  200.0 ml
0.2 M sodium acetate (FW 82.03)          0.82 gm                 3.28 gm
50 mM L(+) tartaric acid (FW 230.1)     0.58 gm                 2.32 gm
Stir using a magnetic stir bar until dissolved;  pH to 5.0

Following the 5-minute wash under running water, incubate the sections in 0.2 M acetate buffer for 20 minutes at room temperature.  After this time has elapsed, to this same acetate buffer add:

0.5 mg/ml naphtol AS-MX phosphate     25.0 mg              100.0 mg
1.1 mg/ml fast red TR salt                        55.0 mg              220.0 mg
Incubate the sections from 1 – 4 hours at 37C, monitoring after the first hour, until osteoclasts are bright red.  Rinse in distilled water, followed by counterstaining in Mayer’s hematoxylin for 1 – 5 minutes (depending on the age of the hematoxylin), wash in running water for 5 minutes, air-dry and mount with permount.
Use 10% EDTA for demineralization for optimal results when doing FFPE tissue.

Ref.  Erlebacher & Derynck J Cell Biol 132:  195 – 210, 1996

September 30, 2003 (Revised)
Hermina Borgerink
Wake Forest University Health Sciences

Hermina M. Borgerink, BA, HTL(ASCP)QIHC
Wake Forest University Health Sciences
Department of Pathology
Medical Center Blvd.
Winston-Salem, NC 27157
Tel. (336) 716-1538
Fax (336) 716-1515

-----Original Message-----
From: [] On Behalf Of Dimaano, Nena
Sent: Tuesday, July 27, 2004 4:52 PM
Subject: [Histonet] TRAP Staining

Hello everyone,
I wanted to make a quick informal survey to see how many researchers/techs are successful using TRAP (histochemistry) staining on formalin fixed, paraffin embedded sections. I would also like to find out those having problems? 
thanks for your help,
Nena Dimaano, MT/HT(ASCP)
Advanced Technology
Stryker Orthopaedics
325 Corporate Drive
Mahwah, NJ 07430
tel: 201-831-5338
fax: 201-831-6224

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