Alexa555 goat anti rabbit igg

Cells were fixed with 4% paraformaldehyde at RT for 15 min, then incubated with blocking buffer (PBS with 5% NCS and 0.2% Triton X-100) for 1 h at RT and with primary antibodies at 4 °C overnight. The proteins were detected with Alexa 647 goat anti-mouse IgG or Alexa 555-goat anti-rabbit IgG (Invitrogen) for 1 h at RT, and this was followed by nuclear staining with Hoechst 33258. Images were captured on a Leica TCS SP5 II confocal microscope (Leica Microsystems, Wetzlar, Germany) with 100× oil objective lenses and a numeric aperture of 1.40 N. Images of the cells were acquired from a 0.13 μm optical section, and no labeling was observed when the secondary antibody was used alone. For the tumor tissue staining, 5 μm slices of the FFPE tissue sections were deparaffinized in xylene and rehydrated through graded ethanol. The sections were incubated with citrate buffer (pH 6.0) in a 95 °C water bath for 20 min for antigen retrieval and then subjected to immunofluorescence staining as described above.