After a rinse in TBS (three times, 5 min) binding sites of primary antibodies were visualized by corresponding AF488, AF555 and AF647 tagged antisera (1 1000; Invitrogen, Karlsruhe, Germany) in TBS, containing 1% BSA and 0.5% Triton X-100 (1 hour at RT) followed by another rinse in TBS (three times, 5 min).
Bound antibodies were visualized by chemiluminescence.
Biotinylated antibodies were visualized with streptavidin-Cy5.
Subsequently, the dot-blots were washed again and binding of antibodies was visualized by alkaline phosphatase conjugate substrate kit (Biorad, Hercules, Canada).
Specific binding of the antibodies was visualized using SuperSignal West Dura Extended Duration Substrate (Pierce, Rockford, IL) after which the blots were exposed to film (Kodak).
The binding of the primary antibodies was visualized using fluorescein labeled anti-goat antibody (1 100, Invitrogen); Alexa Fluor 488 labeled anti-rabbit (1 200, Invitrogen); and Alexa Fluor 594 labeled anti-mouse (1 1000 or 1 400, Sigma Aldrich and Invitrogen respectively).
Subsequently, binding sites of the primary antibody were visualized using a Dako EnVison kit (Dako, Glostrup, Denmark) according to the manufacturer's instructions.
After three 10 min washes in TTBS, binding of primary antibody was visualized using horseradish peroxidase-conjugated secondary antibodies, and the immunolabeling was detected by an enhanced chemoluminescence (ECL) method according to the manufacturer's instructions (Pierce Chemical Company, USA).
The binding of the specific antibody was visualized using Fuji film LAS-3000 (film film) after treating with enhanced chemiluminescence system reagents (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
