Tcs sp5 x confocal laser scanning microscope

Cells were cultured in 12-well plates. On days 11, 21 and 29 after induction, cells were fixed in 4% paraformaldehyde (Sigma) for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 for 15 minutes at room temperature, blocked in 3% bovine serum albumin for 30 minutes at room temperature, and then incubated overnight with primary antibodies at 4°C. The primary antibodies used (all from Millipore, Temecula, CA, USA; all diluted 1:1000; mouse anti-human) were: class III β-tubulin (Tuj-1), microtubule-associated protein 2 (Map2), neuron-specific nuclear protein (NeuN) and neural cell adhesion molecule (NCAM). The following day, cells were incubated with goat anti-mouse AlexaFluor 488 or 594-labeled secondary antibodies (1:1000; Invitrogen) for 1 hour at 37°C. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; 1:5000; ZhongShan Goldenbridge Biotechnology Co., Ltd., Beijing, China) for 5 minutes at room temperature. Morphology was examined using a Leica TCS SP5 X confocal laser scanning microscope (Wetzlar, Hesse, Germany). Experiments were repeated five times. At least 10 immunofluorescence staining pictures were taken per experiment, and all the Tuj-1-positive cells were counted. The reprogramming efficiency was expressed as Tuj-1-positive cells/total cells.