Sections of the paraffin-embedded cell pellets and the breast cancer tissues were prepared and processed for antigen retrieval as above. They were processed for IHF as described previously [13 (link)]. After being blocked with 10% goat serum for 20 min at room temperature, they were incubated with anti-γ1-adaptin antibody (BD Bioscience; 1:1000) alone for cell pellets, or with a mixture of anti-γ1-adaptin antibody and either rabbit anti-EEA1 antibody (Abcam, Cambridge, UK; 1:200 dilution) or sheep anti-human TGN46 (Bio-Rad Laboratories, Inc., CA, USA, 1:400 dilution) for patient specimens, for 1 day at 4 °C. They were then incubated with Alexa 594-conjugated donkey anti-mouse IgG (1:800 dilution), or a mixture of Alexa 594-conjugated donkey anti-mouse IgG and Alexa 488-conjugated donkey anti-rabbit IgG for 60 min at room temperature. They were observed with a confocal laser microscope (FLUOVIEW FV1000, OLYMPUS, Japan).
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