Dcs e995

SB discs were fixed in a neutral buffer containing 4% formaldehyde, washed, and decalcified with MicroDec EDTA-based from Diapath. Specimens were then dehydrated and paraffin-embedded through EZ Prep Concentrate solution (Ventana Medical Systems Inc.). Sections were stained for H&E for morphological analyses. Immunohistochemical analysis (IHC) was performed by the automated instrument BenchMark ULTRA (Ventana). Tissue sections were incubated with the following primary mouse monoclonal antibodies (MoAb) from Abcam: COLL-1 (ab34710, at 1 : 400 dilution), OCN (ab93876, at dilution 1 : 250), and TGFβ (ab92486, at dilution 1 : 150). They were titrated to yield maximal specific staining and minimal nonspecific or background staining. The endogenous peroxidase activity was inhibited by the addition of ultraView Universal DAB Detection Kit (Ventana). All samples were counter-stained with Mayer's hematoxylin solution (Roche) and mounted with Kaiser's glycerol gelatin. Slides were examined double blind, and microphotographs were taken using an Olympus BX51 microscope equipped with a digital camera (Nikon DCS E995).

After removing all medium traces, SB and SBlyo samples were fixed in a neutral buffer containing 4% v/v formaldehyde, washed, and decalcified with MicroDec EDTA-based solution by Diapth. Specimens were then dehydrated and paraffin-embedded through EZ Prep Concentrate solution, cut into thin sections, and stained with hematoxylin and eosin (H&E) for morphological analyses. Immunohistochemical analysis was performed using the automated instrument BenchMark ULTRA (Ventana, Milan, Italy). Tissue sections were incubated with the following primary mouse monoclonal antibodies, purchased from Abcam, Cambridge, UK: OCN (ab93876, at 1:250 dilution), transforming growth factor-β (TGF-β) (ab92486, at dilution 1:150), and COLL-1 (ab34710, at 1:400 dilution). Each monoclonal antibody was titrated to yield maximal specific staining and minimal nonspecific (or background) staining. The endogenous peroxidase activity was inhibited by the addition of ultraView Universal DAB Detection Kit. All samples were counter-stained with Mayer’s hematoxylin solution and mounted with Kaiser’s glycerol gelatin. Slides were examined double-blind, and microphotographs were taken using an Olympus BX51 microscope equipped with a digital camera (Nikon DCS E995).