Immunofluorescence and TUNEL staining

Paraffin sections were stained with a pH3 antibody (rabbit polyclonal, Cell Signaling Technology, 9701, 1:400), anti SOX9 (Sigma-Aldrich, HPA001758, 1:200), anti Col2A1 (Developmental Studies Hybridoma Bank, CIIC1-b, 1:250) and anti-Myosin II (Developmental Studies Hybridoma Bank, CM1123, 5 µg/ml). Standard antigen retrieval with DIVA Decloaker (BioCare Medical) was carried out. Species-specific secondary antibodies tagged with Alexa Fluor 488 or Cy5 were used (goat anti-mouse 488, A11029; goat anti-rabbit 488, A11034; goat anti-mouse Cy5, A10524, Life Technologies; all at 1:250).

Apoptosis was assessed by TUNEL analysis using the ApopTag Apoptosis Kit (Millipore, S7111) and was detected using fluorescein-tagged anti-digoxygenin antibody as described by Hosseini-Farahabadi et al. (2013). A qualitative assessment of presence of TUNEL-positive cells was carried out.

Nuclei were counterstained in Hoechst 33258 (Sigma-Aldrich, 10 µg/ml in PBS) for 30 min. Slides were mounted with Prolong Gold without DAPI (Invitrogen, P36934). Fluorescence imaging was conducted using a 20× objective on a Panoramic MIDI II slide scanner with 488, Cy5 and DAPI filter sets (3D Histech). High-magnification images were captured with CaseViewer software v.2.4.0.119028 (3D Histech).