Alexa 488 conjugated goat anti mouse f ab 2

HeLa cells expressing hERG were cultured on glass cover slips. Cell-surface hERG was labelled with monoclonal anti-HA Ab (1:1000, on ice) prior to fixation with 4% paraformaldehyde (15 minutes at room temperature). Cell-surface Ab-hERG complexes were labelled with Alexa-488 conjugated goat anti-mouse F(ab′)₂ at room temperature (1:1000; Molecular Probes). hERG post-endocytic fate was visualized using an anti-HA Ab capture assay. Early endocytic hERG pools were labelled by continuous anti-HA Ab capture for 15 minutes at 37 °C. The cell surface hERG pool was then blocked with mouse monovalent F(ab′)₂ fragment on ice (1:100; Jackson). The internalized hERG pool was chased for the indicated time at 37 °C prior to fixation with 4% paraformaldehyde. Cells were permeabilized with 0.05% saponin and intracellular Ab-hERG complexes fluorescently labelled with Alexa-488 conjugated goat anti-mouse F(ab′)₂ at room temperature (1:1000; Molecular Probes). Lysosomes were labelled with polyclonal rabbit anti-LAMP1 Ab (1:1000; Abcam) and Alexa-555 conjugated goat anti-rabbit F(ab′)₂ (1:1000; Molecular Probes). Confocal images were taken on a LSM780 microscope (Carl Zeiss MicroImaging) equipped with a Plan Apochromat 63x/NA 1.4 objective in multitrack mode. Representative single optical sections are shown. Antibodies used for immunostaining have been previously described^{37 (link)}.