Immunocytochemistry

TERA-2.cl.SP12 cells were seeded at 5000 cells per well on poly-d-lysine (25 μg/ml) coated cover slips 22 × 22 mm, high precision (170 ± 5 μm) in 6-well plates. At the end of the experiment, cells were fixed in 4% para-formaldehyde (PFA) in PBS for 30 min at room temperature and rinsed with PBS. For intracellular staining, cells were permeabilised with 1% Triton-X-100 (Sigma) in PBS for 10 min at room temperature. Non-specific labelling was blocked by incubation for 1 h at room temperature with 1% goat serum (Sigma) in PBS with 0.2% Tween-20 (Sigma). Primary antibodies were diluted in blocking solution and incubated with cells for 1 h at room temperature with a β-III tubulin antibody (TUJ-1) 1:200 (Affymetrix eBioscience) or CK-8 antibody 1:500 (Affymetrix eBioscience). After washing with PBS, cells were incubated for 1 h in the dark with anti-mouse FITC-conjugated secondary antibody IgM 1:128 (Sigma) for A2B5 staining or anti-mouse Alexafluor 488 IgG 1:600 (Invitrogen) for TUJ-1 and CK-8 staining. Hoechst 33,342 nuclear staining dye (Molecular Probes) was used at 1:1000 in blocking solution after the secondary antibody step. Fixed and stained cells were visualized using a Leica SP5CLSM FLIM FCCS confocal microscope.