Rhodamin conjugated phalloidin

Cultured cells were xed with 4% paraformaldehyde (Electron Microscopy Sciences, Hat eld, PA) in PBS (pH 7.2) for 15 min at room temperature, then permeabilized in 0.1% Triton X-100 in PBS for 10 min at 4°C. Nonspeci c binding sites were blocked with 2% bovine serum albumin (BSA; w/v) and 5% (v/v) heatinactivated goat serum in 0.05% Triton X-100 in PBS (PBT-1) for 30 min at room temperature. The cells were then incubated overnight at 4°C with primary antibodies diluted in PBT-1: nestin (1:100; Santa Cruz Biotechnology, Inc., CA), myosin VII (1:100; Santa Cruz Biotechnology), Math1 (1:100; BioVision, Mountain View, CA), and Tbx1 (1:200; Invitrogen, Zymed Laboratories, CA). The following day, unbound antibodies were removed by three 15-min PBT-1 washes and one 15-min wash with PBT-1 lacking BSA or goat serum (PBT-2). Cells were then incubated overnight at room temperature with uorophore-conjugated secondary antibodies in PBT-2 (1:200). After three 15-min washes with PBT-2, DAPI was used to visualize cell nuclei.
For some specimens, counterstaining was performed with rhodamin-conjugated phalloidin (Invitrogen) to visualize lamentous actin. Control groups consisted of spheres incubated without any primary antibody. In all experiments, no speci c immuno uorescence labeling was detected in control spheres incubated without primary antibody.