Osteodec

Femoral specimens (n = 5 per group) were fixed in 10% formalin for 24 hours. The bones were decalcified in Osteodec (Bio-Optica, Italy) for 7 days and dehydrated in alcohol scale before embedding the specimens in paraffin and cutting into 5 μm longitudinal sections. The slides were stained with haematoxylin and eosin (H&E) to assess morphology and with Gram staining for bacterial examination. Photomicrographs were captured using an Olympus IX71 light microscope and an Olympus XC10 camera (Japan). The samples were evaluated by two blinded observers to assess the percentage of the fracture healing and signs of osteomyelitis according to a grading score proposed in our previous study [31 (link)]. The Gram positive staining was evaluated as present or absent.

Stifle joints were carefully dissected. Medial and lateral sections of the femoral condyles and tibia plateau were obtained using a band saw. Additionally, a synovial membrane fragment adjacent to the patellar ligament was obtained from each joint (42 (link)). Samples were decalcified (Osteodec, Bio-Optica, Milano, Italy), paraffin-embedded, and sectioned at 6 μm using a microtome (Leica RM 2255, Leica Biosystems, Wetzlar, Germany). The slides were stained with hematoxylin and eosin (H-E). All the sections were captured using a motorized stage light microscope and a PC-based image capture system (BX51, DP71, Olympus Corporation, Japan). The severity of OA lesions were graded histologically using an adapted scoring system from Osteoarthritis Research Society International (OARSI) (33 (link), 43 (link)) (Table 1). Two independent and experienced observers blinded to the treatments performed the scoring.

Femurs (n = 6 per group; n = 3 s-rBMSCs) were fixed in 10% formalin, decalcified in Osteodec (Bio-Optica), embedded, and cut into 5 µm sections. Haematoxylin and eosin (H&E) staining was performed to assess morphology, fracture healing, and signs of osteomyelitis. The Gram-positive staining was evaluated for presence or absence of bacteria. Olympus IX71 light microscope and Olympus XC10 camera (Japan) were used to obtain images.

Bone marrow biopsies (BMBs) from our myeloma patients were fixed in Schaffer fixative for 24 hours and decalcinated in osteodec (Bio-Optica, Milan, Italy) for 4-5 hours. Sections were stained with hematoxylin-eosin, Giemsa, periodic acid Schiff (PAS), Prussian blue, and Gomori's stain for reticulin fibers. Sections of paraffin embedded BMB samples were processed for immunohistochemical analysis in a Dako Autostainer Plus (DakoCytomation Colorado, Fort Collins, CO, USA) according to the manufacturer's protocol using the Envision procedure (DAKO EnVision FLEX, High pH KIT K801021, Glostrup, Denmark). Samples were routinely immunohistochemically stained with anti-CD138 (clone MI15, m7228, DAKO Glostrup, Denmark), Ig kappa (number 40191, DAKO, Glostrup, Denmark), and Ig lambda (number 40193, DAKO, Glostrup, Denmark) antibodies for detection of the monoclonal antibody anti-CD34 Class II (m7165 clone QBEnd10, DAKO, Glostrup, Denmark), which was used to highlight endothelial cells. Epitope retrieval was achieved by immersing slides in Tris-EDTA buffer (pH 9.0) and boiling for 15 minutes in a water bath at 97°C. The slides were then incubated with CD34 monoclonal antibody at 1 : 100 dilution for 30 minutes at room temperature. For negative controls, a limited number of cases were stained by substituting primary antibody with buffer solution (DAKO).

Both SGs and the pseudocapsule were fixed in 10% buffered formalin, decalcified for 48 h in Osteodec (Bio-Optica S.p.A., Milan, Italy), embedded in paraffin, sectioned at 4 μm and routinely stained with haematoxylin and eosin. A histological diagnosis was reached by the pathologists of the Department of Veterinary Science, University of Turin.