Hello everyone. We have FFPE decalcified mouse tibiae, and would like to stain for osteoid, woven and lamellar bone. Can you suggest a staining protocol that would enable us to distinguish between these types of tissue? Also, if anyone would be so kind to share a recipe, that would be appreciated. I did find a reference to a modified tetrachrome method by Ralis and Watkins, however, since I do not have access to their full text from 1992, I also have no recipe. Some help with this or another method would be great. Thanks in advance!
I am not an expert as far as stains are concerned, but I just wanted to share that osteoid can be distinguished very well by immunohistochemistry using Anti-Biglycan antibody.
I am not aware of a specific stain that will distinguish between lamellar and woven bone. The distinction is usually made by examination of the structure of the tissue using standard stains, like H&E. Examining the stain under polarized light is also useful, as it will make more prominent the lines of new bone formation or resorption. Similarly, osteoid is usually identified by its appearance (amorphous) on standard stains.
As far as osteoid contains mainly of cartilage and collagen alcianblue (for proteoglycane) and PAS (for collagen fibers) can highlight this structure. Bone-structure can be shown with reticulin-stain.
http://library.med.utah.edu/WebPath/HISTHTML/MANUALS/MANUALS.html
Dear everyone, thank you for your helpful advice. Ms Lang, thanks for the link, I will share it with my colleagues, it seems very handy. Best regards!
We work with undecalcified bone samples embedded in PMMA and we stain with toluidine blue pH 3.7 and it allows to distinguish clearly between calcified bone (blue) and osteoid (pale pink). As it is stated by jean-Martin Lapointe, to distinguish between lamellar and woven bone, an examination of the tissue structure is needed.
Regards.
Hi Nikola,
I have the same problem that i don't have excess to this paper of Ralis and Witkans. I am now going to perform another staining instead, i gonna use Movat's pentachrome staining which also differentiates between woven and lamellar bone. Another option is Attwood's staining.
What did you got to use in the end and what was your experience?
Kind regard
Hello Jasper.
Unfortunately, we had to postpone our tibia staining for now due to different reasons. However, methods you decided to use seem quite interesting. Would you care to post here when you have some results and which method would you recommend from your experience? Thanks.
Best regards
Hi Nikola,
That's indeed unfortunate. But sure I will keep you updated about my results and experience with this stainings.
Best regards
The simplest way to differentiate woven bone from lamellar bone is by viewing the H&E section using transmitted polarized light as mentioned by Jean-Martin Lapointe. The lamellar bone will have a very organized appearance whereas woven bone will have a haphazard appearance. I use an old pair of broken sunglasses with polarized lenses, placing one between the light source and the substage condenser and the other between my eye and the ocular. Osteoid stains pink on H&E. The motto of our laboratory was "We never make mistakes, only inadvertent discoveries."
Dear William
Thank you for your answer. Impressive inadvertence, if I may say so! :)
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