Antigen-purified primary antibodies are recommended. Unpurified rabbit serum or total IgG preparations may contain considerable amounts of unspecific IgGs that will bind plenty of the rbFab-CpH resulting in a lower overall labelling efficiency.

The pH sensitive dye only regaines its fluorescence after the acidification of the targeted cellular compartment.ICC:Pre-incubate the primary antibody with a 2.5-fold excess of Fab-CpH conjugate (e.g., 2 µg of primary antibody with 5 µg of Fab fragments) in a total volume of 10 µl for 30 min at RT in the dark.Adjust the antibody to the desired dilution and immediately use it for your internalization assay.