Sc 1226

To analyze protein expression in the growth plate, immunohistochemistry was performed in serial sections of femur growth plates with minor modifications to what has been described before²⁰ . Briefly, after deparaffinization and rehydration, antigen retrieval was performed in sodium citrate buffer (10 mM pH 6.0) for 15 min at 75 °C. After retrieval, the slides were blocked with 2% serum (horse and goat) followed by incubation overnight at 4 °C with primary antibody (1:100). Sections were incubated with anti-caspase 3 antibody (sc-1226; Santa Cruz Biotechnology, Dallas, TX, USA), anti-proliferating cell nuclear antigen (PCNA) antibody (ab-18197; Abcam, Cambridge, United Kingdom), anti-collagen X antibody (ab-58632 Abcam, Cambridge, United Kingdom) and anti-Ihh antibody (sc-1196; Santa Cruz Biotechnology). Sections were incubated for 1 h at room temperature with corresponding secondary antibody (1:300, BA-9500 Vector Laboratories; 1:500, ab 97,049 Abcam) followed by incubation with an avidin-peroxidase complex (Vectastain ABC-kit PK-6100) and visualized with 3,3' diaminobenzidine (DAB) (Dako K3468) development for 2–3 min. The slides were counterstained with Alcian Blue and dehydrated. Image J software (NIH) was used to quantify stained area and percentages of immunopositive cells in the growth plates.

Pancreata were frozen in OCT compound or fixed with 2% paraformaldehyde. Sections were stained with goat anti-human caspase-3 antibody (sc-1226; Santa Cruz Biotechnology) or rat anti-mouse CD8 antibody (YTS169).