Am I correctly identifying the osteoclasts in my TRAP stains? (pics included)

[Mr. Gunn]

Decalcified FFPE sections of SCID mouse bone were stained with the Sigma TRAP stain kit for 3-4 hours and counterstained with a light regressive methyl green.  I'm seeing some things that are almost certainly osteoclasts, but I'm also seeing clusters of things that have the same morphology and staining pattern, but aren't where I would expect an osteoclast to be.  I'm guessing they might be macrophages, but it would be a big help if someone could look at some of the included pics and let me know if I'm right.  The characteristic features of an osteoclast are, as I understand it, large size, granular cytoplasm, multiple nuclei, location in resorption pits, and accumulation of purple dye in a TRAP stain.  Some of these guys meet all but the location criteria, and I don't know if these are wandering osteoclasts, or if they're more likely macrophages.

10x - There a cluster of granular purple cells to the left of the bone. Are these osteoclasts or macrophages?

TIFF

10x - There's a similar cluster here, to the left, and another set lining the channel filled with yellow RBCs.  Are either group of these osteoclasts?

TIFF

40x - Here's a closeup of the granular purple objects.

TIFF

40x - Here's another section.  I looked at this area before counterstaining and the purple cells here are definitely TRAP⁺.

TIFF

100x - Here's the 100x of the guys above, where you can see that they look similarly granular to the putative macrophages.

TIFF

You can click on the TIFF link below each pic to get a really huge 5 MB TIFF.  Would you agree that pics 3 and 4 are almost certainly osteoclasts, but 1 and 2 might be macrophages, or is there just not enough info to make the determination?

Thanks a great deal for any and all help!

[richard03]

It looks like macrophage, but also looks like mast cell as methyl green will stain mast cell positively.

Histochemical staining (like TRAP) will only give you general image of cell types, but you may need to do immunohistochemistry to confirm. Cathepsin K may be more specific antibody marker for osteoclasts.

Or you can try to do double staining with a macrophage marker, such as CD68 to confirm it.

This page lists most of cell type markers and should be very useful.

http://www.antibodybeyond.com/reviews/reviews.htm

[Mr. Gunn]

It looks like macrophage, but also looks like mast cell as methyl green will stain mast cell positively.

Histochemical staining (like TRAP) will only give you general image of cell types, but you may need to do immunohistochemistry to confirm. Cathepsin K may be more specific antibody marker for osteoclasts.

Or you can try to do double staining with a macrophage marker, such as CD68 to confirm it.

This page lists most of cell type markers and should be very useful.

http://www.antibodybeyond.com/reviews/reviews.htm

Thanks very much for your help, Richard.

I know they aren't mast cells, because I looked at the slides before the methyl green.  Whatever they are, they're TRAP⁺, and I don't so much need an absolutely positive ID, as I need a method that will allow me to measure the effects of a drug on bone resorption, so some false positives are OK.  One thing I don't know that would be helpful is the average expected amount of osteoclasts in a section of normal mouse bone, because even if all of the guys above were osteoclasts, I don't know if I'm seeing more or less that I should.  See, these sections are from normal animals, and are intended to be my reference group/positive controls, but I need to know if they're actually looking like they're supposed to, first! 

[Mr. Gunn]

I guess I could go two ways:

If I could obtain a bone from a osteoclast deficient mouse(anyone worked with this one or this one?) which didn't have a macrophage defect, then I could stain the sections and if I didn't see a bunch of staining, I could assume the staining in my animals is probably mostly osteoclasts.

The other way would be to try to get the IHC worked out using Cathepsin K or CD68.  Can anyone recommend an antibody and protocol for these?  I'll repost this in the IHC forum, also.

I've had problems getting any kind of IHC to work well on these FFPE, decalcified sections(bone glows under a variety of fluorescent excitations), so I would rather not go there if I could choose option 1, but perhaps I should try both.

[ImmunoNYC]

The cells in the last two photos looks like osteoclasts to me. They are in the place you would expect them, next to bone.

[Mr. Gunn]

Thanks for the reply.   I am going to see if counting only the osteoclast/macrophage objects which are located next to bone gives the expected results in my control samples.  If it does, I can probably get the answer I'm looking for without having to resort to laborious immunostaining.

[Mr. Gunn]

So I've been working on a method, and I think I've got it mostly figured out.  I took the inspiration from this paper in JHC.

Here are some pictures.

Green = ELF97
Blue = cathepsin K
Red = PI