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
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