Fluorescently conjugated donkey anti rat ig

B cells were activated for 20 h with anti-CD40 + IL-4, transferred to anti-CD44-coated glass cover slips and incubated for an additional 22 h. They were subsequently fixed, washed, permeabilized, treated with anti-mouse CD16/CD32, and incubated with rat anti-mouse α-tubulin antibodies (Abcam). After washing, cells were further stained with fluorescently conjugated donkey anti-rat Ig (which had minimal cross-reactivity with mouse Ig; Jackson ImmunoResearch), fluorescently conjugated phalloidin (Sigma-Aldrich) and Hoechst 33258 (Sigma-Aldrich). Cells were observed and images were captured using a Leica DMLB Fluorescent Microscope, with an HCX PL APO 63×/1.32 oil lens, DC350F CCD camera, and IM500 Software (Leica Microsystems).

Stimulated B cells on glass coverslips were fixed, washed, permeabilized, treated with anti-mouse CD16/CD32, and then stained with fluorescently conjugated phalloidin, Hoechst 33258 (both from Sigma-Aldrich), rat-anti-mouse α-tubulin (Abcam), and fluorescently conjugated donkey-anti-rat Ig (minimal cross-reactivity to mouse Ig, Jackson ImmunoResearch). Cells were observed, and images were captured using a Leica DMLB Fluorescent Microscope, HCX PL APO 63×/1.32 oil lens, DC350F CCD camera, and IM500 Software (Leica Microsystems).