Mrc600 laser scanning confocal microscope

Cells cultured on gelatin-coated coverslips were washed in PBS and fixed in 3.7% formaldehyde for 15 minutes at room temperature. After blocked with 10% goat serum for 15 minutes, the coverslips were incubated with primary antibody for 60 minutes and fluorescently labeled secondary antibodies for 45 minutes at 37°C successively, and then washed extensively and mounted. Cells were viewed by use of a Bio-Rad MRC 600 laser scanning confocal microscope.

The cellular localization of ERα and ISG12 was determined by indirect immunofluorescence microscopy. Briefly, MCF-7 cells were grown on glass coverslips and fixed with freshly prepared 2% paraformaldehyde solution. The cells were incubated first with primary antibodies and then with secondary antibodies conjugated with Alexa-546 (red) and Alexa-488 (green; both from Molecular Probes, Eugene, OR). Prolong-Gold Antifade reagent with DAPI (blue; Invitrogen) was used to counterstain the DNA. Confocal analyses were performed using the Leica TCS SP8 confocal microscopy system and MRC600 laser-scanning confocal microscope (Bio-Rad, Hercules, CA). Each slide was examined at three excitation wavelengths (488, 546 and 633 nm). Quantification of nuclear ERα immunofluorescent signal (ERα signal/area) in control MCF-7 and MCF7-ISG12 cells is represented as mean ± SE. of three independent experiments (25–120 nuclei, each). Statistical significance (p value) for differences between MCF-7 and MCF7-ISG12 cells is shown as p < 0.05.