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Biotinylated antibodies were visualised by secondary staining using FITC-, PE- or Tricolor-streptavidin conjugates (Caltag).

4LO3311 supernatant was used and secondary stained using an anti-mouse IgG3 PE-Cy7 (Southern Biotech).

Followed by secondary staining with HRP-conjugated goanti-humanmantibodyody (1∶2,000).

SO globes were labeled using polyclonal rabbit antinitrotyrosine (primary antibody)and sheep-antirabbit IgG conjugated with flourescein (secondary antibody), followed by TUNEL staining using a TACS TdT Apoptosis Detection Kit (American Qualex).

Proteins were detected by staining using appropriate secondary antibodies.

The expression of PVR/CD155 on MM cells was analyzed by immunofluorescence staining using an anti-PVR/CD155 unconjugated mAb, followed by secondary GAM-APC.

Primary and secondary antibody incubations were subsequently undertaken at 37°C for 1 hr each followed by nuclear staining using DAPI (1 µg/ml) diluted 1/500 in water.

Blots were exposed to the corresponding horseradish peroxidase-conjugated secondary antibody (Promega) and developed by enhanced chemiluminiscent staining using ECL western blotting system (Amersham Biosciences).

We confirmed this differential staining using secondary anti-IgG antibodies alone (not shown).

Briefly, after approximately 5 days of growth, small plugs of biofilm were taken using a pipette with the tip cut off and then were stained by secondary immunofluorescence using an anti-HA primary antibody.

Secondary staining was performed using the EnVision + System (HRP Labelled Polymer Anti-Rabbit K4003, DakoCytomation).