Mielodec

The bones were fixed in 10% neutral buffered formalin, decalcified in Mielodec (Bio-Optica, Milan, Italy) and embedded in paraffin; serial sections (4 μm) for each specimen from three mice per group were stained with hematoxylin and eosin to verify bone metastasis presence. After antigen retrieval (95 °C for 20 min at pH 6 in antigen-unmasking solution, Vector Laboratories, Burlingame, CA, USA), sections were treated for 10 min with 0.1% (v/v) H₂O₂ and blocked with normal serum. Immunostaining was performed on bone specimen slices from three mice with anti-Twist (1:200), anti-Snail (1 µg/mL), anti-human HGF (3 μg/mL) or anti-mouse HGF (10 μg/mL) antibody, using a streptavidin-biotin system (ABC kit, Santa-Cruz Biotechnology) and diaminobenzidine substrate, and counterstaining with Meyer’s haematoxylin [25 (link)]. Negative-control sections were subjected to the same staining procedure without the primary antibody.

Femoral specimens (n = 4 per group) were fixed in 10% formalin overnight, then decalcified in Mielodec (Bio-Optica, Milan, Italy), dehydrated, the metallic implants were removed, samples were embedded in paraffin, and cut into 5 μm sagittal sections. After deparaffinization, the slides were stained with Haematoxylin and Eosin (H&E) and Gram staining to assess the presence of bacteria, finally analyzed by three independent examiners using an Olympus IX71 light microscope. The inflammatory response and infection were both evaluated trough the periosteal reaction, cortical bone and medullary canal changes according to the grading score (0 to 3) described in our previous study [24] . Briefly, the periosteum was analyzed for absence or presence of reaction; the cortex was mainly analyzed for absence or presence of polymorpho-nucleated cells, osteoclasts and bone resorption; and the medullary canal for absence or presence of polymorpho-nucleated cells and micro- and macroabscesses.