Hello everyone, I was just wondering if someone could offer some advice or point me in the direction of a paper that could answer my question?
We currently have some femur samples that have been collected from pigs, and I wish to do some histological TRAP staining in order to observe osteoclast cell numbers. I'm aware that the general procedure is to fix the tissue and decalcify before any staining is done, but I want to be very careful in how I actually sample the bone before fixing. There are questions such as: what would be the best method to cut a sample from the femur prior to fixing, and should TRAP staining be done on the surface of the cortical bone, or should we aim for staining a cross-section of the bone?
Therefore can I ask if anyone has prepared whole bone samples for histology before, or knows of any good papers that could point me in the right direction?
Many thanks in advance.
Hello Adam,
If you work on undecalcified or decalcified bones, you must first do tissue fixation (then decalcification if it’s the chosen method), then embedding in paraffin (decalcified bone) or methylmetacrylate (undecalcified bone) and the block can then be sliced and processed (TRAP or other staining). Both plastic (methylmethacrylate) and paraffin embeddings work for TRAP staining.
Large animal bones should be prestrimmed to an approximate thickness of 3-4 to 10 mm using a high-quality saw, with a section at the center of the diaphysis or where the area of interest has been predetermined. To protect the bone cells from damage caused by decalcifying agents, it is imperative to fix the samples correctly before decalcification, even lengthening the fixation times. To allow fixation agents access to bone, skin and soft tissues must be carefully removed.
References
1- Chappard D, Palle S, Alexandre C, Vico L, Riffat G. Bone embedding in pure methyl methacrylate at low temperature preserves enzyme activities. Acta Histochem 1987;81:183-90.
2-Helfrich M., Ralston S. (eds) Bone Research Protocols. Methods in Molecular Biology (Methods and Protocols), vol 816. Humana Press, Totowa, NJ
https://doi.org/10.1007/978-1-61779-415-5_19
3- An Introduction to Decalcification, Author: Geoffrey Rolls
https://www.leicabiosystems.com/pathologyleaders/an-introduction-to-decalcification/
Hi Adam:
Whenever possible, we do not decalcify bone. We leave the mineral intact, because it gives integrity to the tissue, because it is highly integrated into the cellular milieu, and because it leaves the sections available for staining techniques and other diagnostic imaging techniques that are able to examine the quality and properties of the mineral phase, such as density-dependent imaging in the SEM. We fix the bone in 10% buffered formalin (the old standby), or 70% ethanol. We then embed the bone in plastic (polymethylmethacrylate), cut the block at the desired level, and make a thin section. We then use a TRAP staining kit compatible with plastic embedded bones by simply surface staining.
Hello Adam,
concerning your question about what would be the best method to cut a sample from the femur prior to fixing:
There are specialized diamond band saws for an application in pathology and anatomy available, produced for instance by EXAKT Advanced Technologies in Norderstedt/Germany. The diamond coated band allows you to cut very gently through the hard mineralized bone without disturbing its structure. This can happen easily in cancellous bone, where trabeculae can fracture if you use serrated saw bands.
For the begining the use of the following band saw, for which diamond coated bands are available can be helpful:
https://www.proxxon.com/en/micromot/27172.php
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