Alexa 546 conjugated anti rabbit igg red color emission

HepG2 cells were cultured and fixed in 4% paraformaldehyde for 10 min, and for membrane permeabilization, we used 1XPBST (containing 0.1% TX-100 detergents and PBS) for 10 min, then washed with 1XPBS and incubated with specified primary antibody (1:100) overnight at 4 °C. After primary antibody incubation, they were again washed with 1XPBS, then p-PDHA1 and PKM2 antibody recognized by an Alexa Fluor 488-conjugated secondary antibody (green-color emission) and Alexa-546 conjugated anti-rabbit IgG (red color emission, Molecular Probes) with 1:50 dilution for 2 h at room temperature. DAPI (1 μg/mL) was also added 10 min before washing to label the nuclei [16 (link)]. For dual immunofluorescence, either monoclonal anti-p-PDHA1 or anti-PKM2 at 1:100 dilution was added and incubated with the cells overnight at 4 °C. Then, we added Alexa-488 conjugated anti-mouse IgG (green color emission, Molecular Probes) with 1:50 dilution and Alexa-546 conjugated anti-rabbit IgG (red color emission, Molecular Probes) with 1:50 dilution, respectively. The nuclear region was stained with DAPI (4’,6-diamidino-2-phenylindole). Fluorescence images were obtained with a conventional fluorescence microscope (Axiovert 200, Zeiss, Oberkochen, Germany). We measured the cyan color intensity in the nucleus by using Adobe Photoshop version 7 and plotted the relative intensity as a bar diagram.