Sp5 2 inverted dmi6000 based confocal microscope

For immunofluorescence analysis, B cells were adhered to coverslips coated with Cell-Tak (BD Biosciences, Franklin Lakes, NJ) after stimulation. Cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.2% Triton X-100 for 5 minutes, treated with 5% BSA for 1 hour, stained with primary antibody O/N at 4C, and stained with secondary antibody at RT for 45 minutes. Nuclei were then labeled with Hoechst 33342 (Life Technologies, Cat #H3570, 4 µg/ml), washed 3 3 5 minutes in 1% BSA in PBS, and mounted in Fluoromount B (Fisher). Images of p65, c-Rel, and Foxo1 localization were obtained with an inverted Leica SP5-II inverted (DMI6000 based) confocal microscope mounted on a Leica DMI4000 microscope (Leica Microsystems, Wetzlar, Germany) and imaging parameters were optimized independently for each channel to maintain fluorescence within the linear range while maximizing intensity resolution. Images analyzed using custom Macros in ImageJ to obtain nuclear intensities of p65, c-Rel, and Foxo1.