SPAM-LOW: [Histonet] TRAP staining kit

Mon Feb 21 13:06:59 CST 2011

I'm in agreement with Patsy.  We have found in our hands that the Sigma Kit will not work on formic acid decaled bone.  It may work on EDTA decaled bone, the reason I say may is that we have gotten variable staining results with EDTA decaled bone.  For mouse knees and ankles we have gotten great results with EDTA decaled and paraffin processing.  We did modify the protocol a bit.  We tried it on EDTA decaled rat ankles and it did not work.  It might be due to the length of time in EDTA, I'm not sure, but bottom line it may not work.  At least that’s what our experience has been.  We also will use the TRAP antibody, we have found that it works in human mouse, and canine so far on formic acid decaled and paraffin embedded samples (and again I think length of time in decal may impact staining, the canine samples were teeth and surrounding soft tissue and it worked ok but not as well).  We have tried this antibody also on EDTA decaled mouse bone and it did not work, not sure why but in this instance we only received blocks so we did not have any control over fixation, decal or processing.  

If you do run into issues there are other markers out there that will stain osteoclasts, but are not entirely specific to them.  We have used the following antibodies;

Cathepsin K - works on formic acid and paraffin embedded in mouse and rat, but it may stain some inflammatory cells also
ED-1 - granted it’s a macrophage marker but it also work nicely to stain osteoclasts in rat bone, but again since it is a macrophage marker you are going to have to read through that.

Good Luck

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, Colorado 80308
office (303) 682-3949 
fax (303) 682-9060
www.premierlab.com

Ship to Address:
1567 Skyway Drive, Unit E
Longmont, Colorado 80504

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Hermina Borgerink
Sent: Monday, February 21, 2011 11:45 AM
To: Patsy Ruegg; 'Kim Merriam'; 'Histonet'
Subject: RE: SPAM-LOW: [Histonet] TRAP staining kit

Hi Kim and Patsy,

This protocol works well on EDTA decalcified FFPE tissue samples.

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.

0.2 M ACETATE BUFFER
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:

                                                                   for 50.0 ml         for 200.0 ml
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 

OR

Add the naphtol AS-MX phosphate and TR salt to freshly prepared and preheated  0.2 M sodium acetate buffer

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.

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

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

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Patsy Ruegg
Sent: Monday, February 21, 2011 12:46 PM
To: 'Kim Merriam'; 'Histonet'
Subject: RE: SPAM-LOW: [Histonet] TRAP staining kit

Kim if you are asking if TRAP enzyme histochemistry works on formalin fixed
paraffin embedded decalcified bone in my experience it does not.  It works
on smears and cytology slides or GMA embedded bone without decal, it may
work on ETA decaled bone but I do not think it works on ffpe slides.  There
is an antibody to TRAP now you could use on ffpe sections by IHC.  What are
you trying to use it for, osteoclasts???

Regards,
Patsy

Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. Ste.215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
www.ihctech.net 
www.ihcrg.org

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Kim Merriam
Sent: Monday, February 21, 2011 7:39 AM
To: Histonet
Subject: SPAM-LOW: [Histonet] TRAP staining kit

Hi All,

Does anyone know if there is a TRAP staining kit available for histology
(and 
who sells it)?  I have found a bunch of them on google, but they are all for

cell culture.

Thanks,
Kim
 Kim Merriam, MA, HT(ASCP)QIHC
Cambridge, MA 

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