[Histonet] Re: TRAP stain in bone (Nicole Collette)

[Silvina Molinuevo]

Hello Nicole!!
In our laboratory we use a kit from Sigma to stain osteoclasts in bone. It uses Fast Garnet and works very well with paraffin sections.
I think that using Naphthol-AS-MX phosphate, Naphthol-AS-BI phosphate it would be suitable. But the important thing is that you have to use a Tartrate buffer to enhace especificity (osteoclastic phosphatases are tartrete resistant).
If you need additional help please feel free to contact me.
Best regards, Silvina

------------------------------

Message: 7
Date: Fri, 30 Oct 2009 15:40:06 -0700
From: Nicole Collette <collette2 <@t> mail.llnl.gov>
Subject: [Histonet] TRAP stain in bone
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <p06230909c711158b3a1c@[128.115.150.224]>
Content-Type: text/plain; charset="us-ascii" ; format="flowed"

Hello, and Happy Friday!

I am puzzled over the wide array of protocols available for TRAP 
staining (Tartrate-Resistant Acid Phosphatase) to stain osetoclasts 
in bone. I have read a few methods papers, and a few papers that use 
it as a method in their analysis. It seems to be more pH dependent 
than reagent-dependent, since I have seen reagents such as 
Naphthol-AS-MX phosphate and Naphthol-AS-BI phosphate being used for 
both acid and alkaline phosphataste stains. I also found one 
reference that says,

"Naphthol-AS-BI-phosphate is a substrate for alkaline (1) and acidic 
(2, 3) phosphatase. After hydrolysis of the non-fluorescent 
Naphthol-AS-BI phosphate by the enzyme the resulting Naphthol-AS-BI 
can be measured by fluorescence methods."

They recommend 0.5mM in 200mM sodium acetate, pH 5.0.

Is it really that simple? Can I stain with this single reagent and 
photograph my sections on the fluorescent scope (given proper 
rinsing, mounting, etc.)? Anyone know what color it is? I got 4 
filters, I'm guessing one should work.

Other protocols call for Fast Violet B, or Fast Garnet GBC, I'm 
guessing this makes the stain either purple or red, but is there some 
other reason for using it for the reaction?

Any recommendations or bad experiences would be much appreciated. I 
am able to do either paraffin or frozen sections, but prefer paraffin 
if possible. I already have Naphthol-AS-MX phosphate, Naphthol-AS-BI 
phosphate, Fast Red TR (does this work like Fast Garnet GBC, as a red 
stain?), and assorted acids and buffers if that makes a difference. I 
am happy to purchase additional reagents that will work. Thanks so 
much for your help and support!

Sincerely,
Nicole Collette
Lawrence Livermore National Laboratory/ UC Berkeley





      Yahoo! Cocina

Encontra las mejores recetas con Yahoo! Cocina.


http://ar.mujer.yahoo.com/cocina/