Mixing homogenizer

Immunoblotting was performed with standard protocols as previously described [21 (link)]. The tissue from the chamber of dorsal window of diabetic and non-diabetic mice was harvested at 24 hrs after the model was established, placed into 4 ml lysis buffer, incubated for 5 min. on ice, and homogenized with a mixing homogenizer (Kinematica AG, Littau, Switzerland). The homogenates were heated at 95°C for 10 min. and centrifuged at 12,000 × g for 10 min. The protein concentration was measured by the BCA method (Pierce, Rockford, IL, USA). Aliquots of 40 μg of each sample were loaded on 15% SDS polyacrylamide gel, subjected to electrophoresis and transferred onto nitrocellulose membrane. The membranes were blocked with 5% non-fat milk in Tris buffer saline containing 0.1% Tween 20 at room temperature (RT) for 1 hr, and then probed with goat polyclonal antibody to SDF-1 (sc-6193; Santa, Cruz Biotechnology, Santa Cruz, CA, USA) at a concentration of 1:500 at 4°C overnight, followed by incubation with horseradish peroxidase–conjugated anti-goat IgG (Zymed, South San Francisco, CA, USA) at a concentration of 1:1000 at RT for 1 hr. The horseradish peroxidase was detected with a chemoluminescence ECL-Plus kit (Amersham Biosciences UK, Little Chalfont, UK). Tubulin was detected as an internal protein loading control.