Peroxidase coupled anti rabbit

In tissue, samples of 15 μg were loaded on 4–12% Bis-Tris SDS-PAGE (Invitrogen) and transferred onto PVDF membranes (Millipore). Membranes were blocked with 5% non-fat dry milk in PBS-Tween 0.1% and incubated against MMP-9 (Millipore, ab19016) at a concentration of 1:1000 overnight at 4°C. GAPDH or β-tubulin was used as loading controls. Detection was carried out using peroxidase-coupled anti-rabbit (Vector) at 1:10000 for 1 hour at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL). Cell media was thawed to room temperature and mixed with 4x SDS at a 1:3 ratio. Samples of 15 μl were loaded on 4–12% Bis-Tris SDS-PAGE (Invitrogen) and transferred onto PVDF membranes (Millipore). Membranes were blocked with 5% non-fat dry milk in PBS-Tween 0.1% and incubated against MMP-9 (Millipore ab19016) at a concentration of 1:1000 overnight at 4°C. Cell count was used for normalization. Detection was carried out using peroxidase-coupled anti-rabbit (Vector) at 1:10000 for 1 hour at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL).