Hi,
I need a staining method for Osteoblasts and Osteoclasts.
Anyone did some staining for bone?
Would love to get all the help I can.
Guy

gsovak said:

Hi, I need a staining method for Osteoblasts and Osteoclasts. Anyone did some staining for bone? Would love to get all the help I can. Guy

OK,
I found the staining protocols that are needed.
A- for Osteoblast, an Alkaline Phosphatase staining.
B- fo Osteoclasts, a Tratarate resisntant acid Phosphatase procedure is needed.
If any one needs the protocols please write me.
Guy

If it is not too much trouble can you put in the protocols here?

Lucy said:

If it is not too much trouble can you put in the protocols here?

OK I will put them over here.
Guy

As promised here are the two protocols:

Alkaline phosphatase (ALP)- For Osteoblast
Freshly prepared incubation medium
consisted of 25 mg of alfa -naphthyl phosphate disodium salt
(Sigma, St Louis, Missouri) and 25 mg of Fast Violet B salt
(Sigma) in 25 ml of 0.05M Tris-HC1 buffer (pH 10.5)
containing 0.025M MgC12. Sections were incubated for
seven minutes at 37°C, rinsed in reaction buffer, postfixed
for 15 minutes in aqueous 10% paraformaldehyde and
mounted with glycerol-gelatin (Sigma).

Tartrate-resistant acid phosphatase (TRAP) - For Osteoclasts .
The incubation medium consisted of :
alfa-naphthyl phosphate and Fast Violet B dissolved in 25 ml
of 0.05M Na acetate buffer (pH 5) containing 0.05M
sodium tartrate (Sigma). Sections were incubated for 60
minutes at 37°C, rinsed in reaction buffer, postfixed and
mounted as above.

If any help is needed
I would be happy to do so.
Guy

You can also use alizarin red stain for calcium and von kossa stain for phospate.

Thats true,
But,
The Alizarine and Von Cossa is more for the Calcium and Phosphate as you said and not for the cells.
You have another nice staining for dofferent stages of calsification. I will look into this if some one would like to know.

could you show me the protocol about staining for different stages of calcification? Thank you.

Because I am doing some research in osteoblasts.

Thank you once more.

Hi
Here is the link to he protocol , you can find many other protocol in our Protocol section.
http://www.scientistsolutions.com/index.php?a=protocol&c=431&p=127
Guy

Thank you for your information.
Do you have a protocol for mineralization nodule staining in primary osteoblasts?

Thanks.

Alizarin red S is the protocol that would enable you to visualise the calcification of the nodules.
This is the same protocol that I gave you the link.
Guy

thanks a lot.

Ask if you need any more help regarding bone IHC.
Guy

Thank you.

For minerlization nodule staining, which solution can be used for fixation in primary osteoblasts?

I would use para-formaldehyde 4% in PBS. 15 min RT.
Wash well with PBS X3