Methods and Services

Two types of bone specimen will be analyzed by the Core: Skeleton from genetically modified animals and long bones injected with prostate cancer cells. In the former case both vertebrae (lumbar vertebrae L2, L3 and L4) and tibiae will be embedded, but only L3 and L4 vertebrae will be initially analyzed. Sectioning and analysis of the tibiae will be performed only at special request and after consultation between the Core personnel and the principal investigator of the project. For tumor cell injected mice, both the injected bone and its non-injected counterpart will be embedded and analyzed.

Processing/Embedding/Staining of Non-Decalcified Bone Specimen

Calcein-labeled (1) skeletons fixed flat in 10% formalin for no more than six hours at 4°C and dehydrated up to 80% ethanol will be provided to the core by project investigators. According to the type of experimental model (bones of genetically modified mice or injected bones), appropriate specimen will be dissected and dehydrated at 4°C in alcoholic solutions up to 100% ethanol. The remainder of the skeleton will be sent back to investigators. Dissected specimen will be processed according to established protocols (2). They will be infiltrated twice 24 hours at 4°C in a media containing 100 ml destabilized methylmethacrylate (MMA), 14 ml nonylphenyl-polyethyleneglycol acetate (NPG) and 0.33 g anhydrous benzoyl peroxide (BPO). Samples will then be transferred in a medium containing 100 ml MMA, 14 ml NPG, 0.55 g BPO and 500 ml N,N dimethyl-p-toluidine 99%. Polymerization will be carried out at 4°C.

Plastic blocks will be trimmed on a grainer/polisher and sectioning will be performed on a motorized Leica RM2165 microtome equipped with tungsten-carbide disposable blades. Particular attention will be given in positioning the blocks to insure that all specimens have the same orientation when sectioned. Seven micrometers sections will be collected throughout the bones of injected mice (sagittal sections) or until the two third of the vertebrae are reached for genetically modified animals or their control littermates. A set of 10 consecutive sections, located in the same plane between animals, will be selected (#1-10) for analysis. Sections #1-3 and 8-10 (7 microns thick) will be stained with the von Kossa reagent and counterstained by the van Gieson method for initial analysis of the basic parameters (3). This procedure stains mineralized bone matrix in black, osteoid in red and the cells medium pink. Sections 4-7 will be cut at four microns thickness and left unstained and stored for further analyses. Upon particular request, and on a case-by-case basis, these unstained sections will be used to perform cell counts (osteoblasts, osteoclasts) and quantify dynamic parameters (BFR).

For quantification of osteoblast numbers, slide # 4 will be stained with toluidine blue according to a standard protocol (4). For quantification of osteoclast numbers slides # 5 will be subjected to an enzymatic assay for detection of tartrate resistant alkaline phosphatase staining (TRAP) (4), an enzyme specifically expressed by osteoclasts in the bone marrow. The sections will then be stained with faded hematoxylin. Slide # 6 will be left unstained for visualization of calcein at the mineralization fronts under fluorescent light. Measurements will be performed in 30-35 fields on one slide per sample. All slides within a sample will be examined for possible abnormalities which are avoided.