Slowfade light antifade kit

Cells were grown in 13 mm sterile coversilps, washed with PBS and fixed with 4% PFA for 15 min at 37 °C. PFA was quenched by incubating the cells for 10 min in 50 mM NH₄Cl₂ in PBS. After washing, cells were permeabilized with 0.1% triton x-100 for 20 min and then blocked for 1 h at room temperature (RT) with 10% BSA 0.1% triton x-100 in PBS. Cells were probed with mouse anti-Myc 1:200 (cat # SC-40; Santa cruz), rabbit anti-Myc 1:200 (cat # CST2278; Cell signaling technology), rabbit anti-TOM20 1:200 (cat # SC-11415; Santa cruz), mouse anti-ATP synthase (Complex-V-β) 1:200 (cat # MS-503; Mitosciences), rabbit anti-Nanog 1:200 (cat # A300–397A; Bethyl), mouse anti-Oct3/4 1:200 (cat # SC-5279; Santa cruz), mouse anti-ESRRβ 1:200 (cat # H6705; R&D systems), goat anti-T brachyury 1:200 (cat # SC-17743; Santa cruz), and goat anti-FOXA2/HNF-3β 1:200 (cat # SC-6544; Santa cruz) Abs. Next, cells were washed with PBS, stained with second Ab conjugated to Cy2 or Cy3 dyes (Molecular Probes, Inc.), washed, mounted with SlowFade Light Antifade kit (Molecular Probes, Inc.), and analyzed by confocal microscopy.

For immunofluorescence studies, cells were seeded 24 h before the experiment in 13 mm sterile glass coverslips (Thermo Scientific). Cells were then fixed with 6% PFA for 15 min at 37 °C. PFA was quenched by incubating the samples for 10 min in 50 mM NH₄Cl₂ in PBS. After washing, cells were permeabilized with 0.1% triton X-100 in PBS for 20 min and then blocked for 30 min at room temperature with 5% BSA, 0.1% triton X-100 in PBS. Cells were then probed with primary antibodies, washed, and stained with secondary antibodies conjugated to Cy2, Cy3, or Cy5 (Molecular Probes, Inc.). After three additional washes with DDW, coverslips were mounted on slides using SlowFade Light Antifade kit (Molecular Probes, Inc.).