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Topic Started by gsovak
on 3/10/2006 14:06 PM
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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
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Replies
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Posted By gsovak
on 3/14/2006 11:59 AM
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| 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
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Posted By Lucy
on 3/14/2006 12:32 PM
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If it is not too much trouble can you put in the protocols here?
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Posted By gsovak
on 3/15/2006 14:06 PM
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| Lucy said: | | If it is not too much trouble can you put in the protocols here? |
OK I will put them over here. Guy
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Posted By gsovak
on 3/16/2006 16:44 PM
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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
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Posted By dawn
on 6/14/2006 17:30 PM
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You can also use alizarin red stain for calcium and von kossa stain for phospate.
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Posted By gsovak
on 6/15/2006 18:54 PM
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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.
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Posted By docqian
on 3/26/2007 10:58 AM
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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.
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Posted By docqian
on 3/28/2007 9:44 AM
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Thank you for your information.
Do you have a protocol for mineralization nodule staining in primary osteoblasts?
Thanks.
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Posted By gsovak
on 3/28/2007 14:18 PM
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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
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Posted By docqian
on 3/29/2007 5:15 AM
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Posted By gsovak
on 3/30/2007 10:23 AM
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Ask if you need any more help regarding bone IHC.
Guy
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Posted By docqian
on 4/2/2007 8:40 AM
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Thank you.
For minerlization nodule staining, which solution can be used for fixation in primary osteoblasts?
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Posted By gsovak
on 4/5/2007 13:29 PM
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I would use para-formaldehyde 4% in PBS. 15 min RT.
Wash well with PBS X3
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