Osteoblast and Osteoclast staining

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Guy Sovak
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Osteoblast and Osteoclast staining

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

Guy Sovak
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gsovak wrote:Hi,

gsovak wrote:

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

Lucy
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If it is not too much trouble

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

Guy Sovak
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Lucy wrote:If it is not too

Lucy wrote:

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

OK I will put them over here.
Guy

Guy Sovak
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As promised here are the two

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

dawn
dawn's picture
You can also use alizarin red

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

Guy Sovak
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Thats true,

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.

docqian
docqian's picture
could you show me the

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.

Guy Sovak
Guy Sovak's picture
Hi

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

docqian
docqian's picture
Thank you for your

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

Thanks.

Guy Sovak
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Alizarin red S is the

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

docqian
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thanks a lot.

thanks a lot.

Guy Sovak
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Ask if you need any more help

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

docqian
docqian's picture
Thank you.

Thank you.

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

Guy Sovak
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I would use para-formaldehyde

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

docqian
docqian's picture
Thank you!

Thank you!

KBA
KBA's picture
gsovak wrote:As promised here

gsovak wrote:

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

Can you please provide me protocol for trap stain in RAW cells osteoclast along with fixing procedure
Thanks

sencajal
sencajal's picture
what will be the role of

what will be the role of osteoblastb and osteoclast.. i think yiou are the right person to ask this question. is that true bone morphogenesis will occur throught the life?

Guy Sovak
Guy Sovak's picture
Hi there,

Hi there,
Osteoblasts are the cells that "produce" the bone,
Osteoclasts "eat" the old bone and make place for new bone to be deposited.
Bone remodeling is a process that happens all our life. Old bone is being absorbed and new bone is being deposited.
Guy

Amywang
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Hey, I am currently working

Hey, I am currently working on osteoblast and osteoclast. please share your protocols for both cells staining. thanks a lot

roseruf
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Hi Everyone!

Hi Everyone!

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We are trying to see mineralization using Alizarin red. It worked very well in the immortalized foetal cell line hFOB1.19, but after 3 weeks, our human primary osteoblasts are not mineralizating.  We are trying to differentiate them with ascorbic acid and glicerophosphate.  Does any one have any experience on this issue? Are 3 weeks enough to see mineralization on human primary osteoblasts? Is this media adequate?
 
And just a silly question: we have seen that we can raise alizarin solution’s pH with amonium hidroxide, can we use NaOH?
 
Thank you very much!

unfazed34695
unfazed34695's picture
 Hi, I was just curious -

 Hi, I was just curious - what is the reaction buffer that these protocols refer to, please?  Thanks a million

thasneem00751
thasneem00751's picture
Hi,

Hi,
i need a staining method for Alkaline phosphatase in decalcified paraffin or resin section. we are having bones implanted  with silica coated hydroxy apatate scaffold containing osteogenic stem cell in goat femoral bone, so i need to study the osteoblastic (ALP) activity in the sections. we already  purchased alkaline phospatase (sigma cat.no-FBS-25) and tried so many times with paraffin sections but i am not getting result. if anybody done it before, plz help me ?