In Situ Hybridization and Histological Staining of Limb Sections

For in situ hybridization (ISH) and histological staining on sections, limbs were processed into paraffin using the Excelsior ES Vacuum Infiltration Processor (Thermo Scientific), embedded in paraffin and sectioned at 5 μm. Non-radioactive ISH were performed using digoxigenin (DIG)-labeled anti-sense RNA probes as previously described (Murtaugh et al., 2001 (link)). Probes for type 2 collagen (Col2a1), indian hedgehog (Ihh), osteocalcin (Ocn), osteopontin (Opn), type 1 collagen (Col1a1), and type 10 collagen (Col10a1) have been published previously (Hill et al., 2005 (link)). The Gfp probe was generated using a plasmid containing the eGFP coding region. All probes are available upon request. Histological stainings such as alcian blue, alcian blue/von Kossa, and hematoxylin/eosin were performed as previously described (Houben et al., 2016 (link)). Skeletal preparations were performed on 6 day old pups which were sacrificed by decapitation, skinned, eviscerated, fixed in 95% EtOH, and double-stained for alcian blue and alizarin red (McLeod, 1980 (link)). The length of isolated humeri and femora was calculated by using Zeiss image analysis software.