Nanozoomer 2.0 digital pathology system

Dissected bones were fixed in 10% formalin and decalcified in formical-4^™ (Decal Chemical Corporation) for 3 days before embedding in paraffin wax [17 (link)]. Bone sections (3–4μm) were stained with Alcian blue 8GX and van Gieson [17 (link)]. For immunohistochemical analysis, dewaxed sections were treated with proteinase K for antigen retrieval, before staining with rabbit polyclonal phospho-p38 MAPK (cat # 4631) and phospho-MKK3/MKK6 (cat # 9231) antibodies (Cell Signalling Technology, Danvers, USA) using the ABC staining system (Santa Cruz Biotechnology, Santa Cruz, USA). Digital images were obtained using the Nanozoomer 2.0 Digital Pathology system (Hamamatsu Photonics, Welwyn Garden City, UK).

Dissected specimens were fixed in 10% formalin, bones decalcified in formical-4 (Decal Chemical Corporation) for 3 days before embedding in paraffin wax. Soft tissue sections (3–4 μm) were stained with haematoxylin and eosin (H&E) or von Kossa, whereas bone sections were stained with van Gieson and alcian blue 8GX, as described [20 (link)]. Digital images were obtained using the Nanozoomer 2.0 Digital Pathology system (Hamamatsu Photonics, Welwyn Garden City, UK). Auditory ossicles were dissected from wild-type and mutant mice and their morphology assessed by microscopy.