Microphot fluorescence microscope

RBCs were fixed with 3.7% formaldehyde in PBS (pH 7.4) for 10 min at room temperature, washed in the same buffer and permeabilized with 0.5% Triton X-100 in PBS for 5 min at room temperature. After washing with PBS, samples were incubated for 30 min at 37°C with monoclonal antibodies: anti-ER-α, anti-ER-β, anti-phosphorylated ERK_1/2 (BD PharMingen, San Diego, CA), anti-survivin (Santa Cruz Biotechnology) and anti-3-nitrotyrosine (Sigma Aldrich). After, all samples were washed thrice in PBS to be then incubated with secondary antibody FITC-conjugated: anti-mouse (Invitrogen, Carlsbad, CA) or anti-rabbit (Invitrogen, Carlsbad, CA). All the samples were recorded with a FACScan flow cytometer (Becton-Dickinson, Mountain View, CA, United States) equipped with a 488 United Statesnm argon laser. At least 20, 000 events were acquired. The median values of fluorescence intensity histograms were used to provide a semi-quantitative analysis. Morphometric analyses were also employed to evaluate ER-α and ER-β distribution. All samples were mounted on glass cover slips with fluorescence mounting medium (Dako) and observed by intensified video microscopy (IVM) with an Olympus Microphot fluorescence microscope (Olympus Corporation, Tokyo, Japan) equipped with a Zeiss CCD camera.

For nuclear detection of Ki-67, control and treated cells were fixed in acetone/methanol 1/1 (v/v) for 10 min at room temperature and air-dried. After 1 h of preincubation with PBS containing 10% of AB human serum, the cells were incubated for 1 h at room temperature with FITC-conjugated mouse anti-human Ki-67 (BD Biosciences). The nuclei were stained with Hoechst 33258 (Sigma-Aldrich) at 37 °C for 15 min. The samples were mounted on glass cover slips with glycerol/PBS (2:1) and observed by intensified video microscopy (IVM) with an Olympus Microphot fluorescence microscope (Olympus Corporation, Tokyo, Japan) equipped with a Zeiss CCD camera.

For nuclear factor kappa B (NF-κB) detection, cells were fixed in acetone/methanol 1/1 (v/v) for 10 minutes at room temperature and air dried. After 1 hour of preincubation with PBS containing 10% of AB human serum, cells were incubated for 1 hour at room temperature with the rabbit polyclonal antibody to NF-κB (Santa Cruz Biotechnology). Following three washes in PBS, cells were incubated for 1 hour at room temperature with FITC-labeled anti-rabbit secondary antibody. Morphometric analyses were also employed to evaluate NF-κB nuclear translocation. The cells with positive nucleus were evaluated by counting 300 cells at high magnification (500x). The nuclei were stained with Hoechst 33258 (Sigma-Aldrich) at 37°C for 15 minutes. For actin filament detection, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 (Sigma-Aldrich), and stained with fluorescein-conjugated phalloidin (Sigma-Aldrich) at 37°C for 30 minutes.
All samples were mounted on glass cover slips with glycerol/PBS (2 : 1) and observed by intensified video microscopy (IVM) with an Olympus Microphot fluorescence microscope (Olympus Corporation, Tokyo, Japan) equipped with a Zeiss CCD camera.