Rabbit anti runx2

Thirty micrograms of proteins obtained from all samples were processed as previously described [29 (link)]. Blotted membranes were incubated with the following primary antibodies: rabbit anti-RUNX2 (1:750, rabbit; Sigma-Aldrich, Milan, Italy), anti-Vimentin (1:750, rabbit; Sigma-Aldrich), anti-Laminin (1:750, rabbit; Sigma-Aldrich), anti-N-cadherin (1:750, rabbit; Sigma-Aldrich) and anti-beta-actin (1:750, mouse; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After five washes in PBS containing 0.1% Tween-20, membranes were incubated for 1 h at RT with peroxidase-conjugated anti rabbit and anti-mouse secondary antibodies (1:2000; ThermoFisher Scientific, Milan, Italy). Protein expression was analysed by the enhanced chemiluminescence detection method (ECL) (Amersham Pharmacia Biotech, Milan, Italy) with photo documenter Alliance 2.7 (Uvitec, Cambridge, UK). Signals were evaluated by ECL enhancing and assessed through an UVIband-1D gel analysis system (Uvitec) [30 (link)].

Primary antibodies: rabbit anti-IHH (1:200, Millipore, Billerica, US); goat anti-PTCH1 (1:50, Santa Cruz, Dallas, US); rabbit anti-GLI1 (1:50, Cell Signalling); rabbit anti-SOX9 (1:50 Santa Cruz); rabbit anti-RUNX2 (1:250, Sigma); PKA phosphorylated substrates (1:150, Cell Signalling); rabbit anti-EDD1 (HsUBR5) (1:100, Bethyl Labs, Montgomery, US). Biotinylated secondary antibodies: goat anti-rabbit and horse anti-goat (1:200, Vector Labs).
Paraffin sections were de-waxed, blocked for endogenous peroxidase and underwent antigen retrieval in 10mM sodium citrate pH6 at 80°C for 30–60 minutes. Slides were blocked with serum-free pan-species block (DAKO, Glostrup, Denmark), incubated with primary antibodies overnight at 4°C, and incubated with biotinylated secondary antibodies for 45mins at room temperature. Sections underwent streptavidin-mediated signal amplification (ELITE ABC, Vectorlabs, Burlingame, US) prior to incubation with peroxidase substrate kit DAB (Vectorlabs).