Hello, I'm trying to do a TRAP staining of mouse femora embedded in paraffin. The bones were fixed for 1 week in 4% buffered paraformaldehyde before they were embedded. After deparaffinization I first incubated the slides in TRAP staining buffer (40mM sodium actetate, 200mM di-sodium tartrate-dihydrat, pH 5,0) for 30min and then in TRAP staining solution (0,5mM Naphthol-AS-MX-phosphate, 3mM Fast Red Violet LB Salt, 2% di-methyleformamide, 1% Triton X-100) for 2,5h. Now I can see a lot of red structures and it looks more like a smear than like osteoclasts. Can anybody help my with the interpretation? Did I incubate the TRAP staining solution too long?
Hi, we are using USEDECALC by Medite but they don't specify their ingredients. I think it contains EDTA but i'm not sure.
Dear Anja,
the TRAP positive structures is true TRAP staining of bone tissue without OCs.
This staining comes from deposited TRAP enzyme within the bone matrix. You know OCs are motile cells that move around the bone surface and secrete vesicles with TRAP. The positive TRAP staining comes from a bone surface that was formerly occupied by OCs.
To evaluate the OC number you need to balance TRAP staining intensity in relation to reliable/surficient visualization of OC cells.
In image 2: the "OC?" is a real OC and the "???" is bone tissue with deposited TRAP enzyme.
If you have further questions please contact me.
All the best,
Jirko
Dear Jirko,
thank you for your answer. I already suspected that it would be deposited TRAP enzyme but I didn't expect it so intensive. That really surprises me and is also very fascinating!
So I try to do a time series to find the best condition of my purpose.
Thanks a lot!
Dear Anja,
of course it suprised me the first time I recognized as well.
Make a time series.
When doing an experiment for quantification try to stain all samples in one experiment. The variability is high. In addition, many other factor affect your result ... decalcification, reliable section thicknes ...
All the Best,
Jirko
Dear Jirko,
doing a time series was very revealing! Half an hour is the maximum for my purpose.
I have a last question: Do you have a explicit publication saying that these TRAP positive structures are deposited TRAP enzyme?
Dear Anja,
congrates to your success.
I am sorry but I have no publication straight in mind. Usually people do not show such images as it initiates questions about quality.
But if I come across such paper I well keep you posted.
All the best,
Jirko
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