Different origins of the phenomena were considered, such as metallic clusters, secondary phases, bound magnetic polarons, charge carriers, and oxygen vacancies [2, 3, 5, 8 13].
The amount of primary and secondary antibody bound to each cell was these examined using a Becton Dickinson FACScan flow cytometer.
After three subsequent washing steps (5 minutes each), the primary antibody was visualized with goat anti-rabbit IgG secondary antibody bound to Alexa Fluor 488 (Invitrogen, Carlsbad, CA) in blocking solution and incubated for 1 hour.
In addition, some primary antibodies may have more than one secondary antibody bound to them.
After rinsing, the sections were incubated with biotinylated secondary antibody bound to a streptavidin-horseradish peroxidase complex.
This could be attributed to variation generated by the length and the spatial orientation of the antibodies (both primary and secondary) that bound heterogeneously to the coatomer.
After a rinse in PBS, sections were incubated 10 min with the primary antibody enhancer, and 1 h at room temperature with the secondary antibody bound to peroxidase.
The horseradish peroxidase (HRP) conjugated goat anti-IgG secondary antibody bound to the monoclonal anti-His antibody was added until enough to cover the membrane.
After rinsing in phosphate-buffered saline, the slides were incubated with a biotinylated secondary antibody bound to a streptavidin-peroxidase conjugate (Vectastain Elite ABC reagent, Vector, Abcys, Paris, France).
After rinsing in PBS, the slides were incubated with a biotinylated secondary antibody bound to a streptavidin peroxidase conjugate (Dako E0468) for 1 hour at room temperature.
