Staining for adhesive structure markers was also performed using monoclonal antibody against vinculin (Sigma-Aldrich, USA) and polyclonal antibody against talin (Santa Cruz, USA) at a dilution of 1 40.
For SP we used an anti-rabbit antibody (Chemicon, Temecula, CA, USA) at a dilution of 1 5000.
For CGRP we used an anti-rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1 3200.
Coverslips were then incubated with 53BP1 antibody (Novus Biologicals, Colorado, USA) at a dilution of 1 1000 in blocking buffer for 1 hour at room temperature.
After washing five times with PBS, the cell-antibody complex was incubated 1.5 h at 4 °C with a goat anti-rabbit IgG conjugated with FITC (Sigma, USA) at a dilution of 1 500.
Primary PLK-1 antibody (Cell Signaling, Technology, Danvers, MA, USA) at a dilution of 1 500 was added to membranes in 5 % BSA (1× TBST + 5 % BSA) overnight at 4 °C.
They were then rinsed three times with washing buffer, 0.1% Triton X-100 in PBS before being incubated with Alexa Fluor 488 Goat anti Rabbit secondary antibody (Invitrogen Molecular Probes, Oregon, USA) at a dilution of 1 1000 in blocking buffer for one hour at room temperature.
The following day, the membranes were washed in TBST and then incubated with HRP-conjugated goat anti-rabbit (GE Healthcare, Piscataway, NJ, USA) at a dilution of 1∶2500 for 45 min at room temperature.
The proteins were then transferred onto PVDF membranes, and probed with anti-AGEs (dilution = 1∶1000, 6D12, Wako, Osaka, Japan) followed by goat anti-mouse horseradish peroxidase (HRP) (KPL, Gaithersburg, Maryland, USA) at a dilution of 1∶2000.
Human IgG antibodies bound to the coated peptides were detected by adding 100 µl/well of phosphatase-conjugated polyvalent anti-human immunoglobulin's (Sigma Aldrich, St Louis MO, USA) at a dilution of 1∶1000.
After washing with phosphate buffered saline, slides were treated with power block reagent (BioGenex, San Ramon, CA, USA) and then with primary monoclonal antibody clone 0211 (AbD Serotec, Raliegh, NC, USA) at a dilution of 1∶10 for one hour.
