4 6 diamino phenylindole dapi

NT2/D1 cells were plated on coverslips and cultured in the absence (for 2 days) or presence (for 2, 4 and 7 days) of RA. Following fixation in 4% paraformaldehyde for 20 min at room temperature and permeabilization in 0.1% Triton X 100, cells were incubated in blocking solution, 10% normal goat serum and 1% bovine serum albumin (BSA) in PBS for 1 h at room temperature. Primary antibodies diluted in 1% BSA (Sigma Aldrich, USA), 0.1% Triton X 100 in PBS were applied overnight at 4°C as follows: mouse monoclonal anti OCT-3/4 (sc-5279, diluted 1: 100; Santa Cruz Biotechnology, Inc) and rabbit polyclonal anti SOX3 (sc-20089, diluted 1: 100; Santa Cruz Biotechnology, Inc). Coverslips were washed three times for 10 min in 0.1% Triton X 100 in PBS and incubated with biotinylated goat anti rabbit IgG (1: 500; Vector, USA) for 1 h at room temperature in 1% BSA, 0.1% Triton X 100 in PBS followed by DyLight 488^® streptavidin (1: 1000; Vector Laboratories, USA) and Alexa FluorH 594, (1: 500; InvitrogenTM) diluted in PBS for 1 h at room temperature. Nuclei were stained with 0.1 mg/ml 4′,6 diamino phenylindole (DAPI; Sigma Aldrich). Samples were viewed and images were taken using a Leica TCS SP8 confocal microscope and Leica Microsystems LAS AF TCS SP8 software (Leica Microsystems, Germany).

3,5-dimethylpyrazole, CuCl, agarose, ascorbic acid, sodium azide (NaN₃), DMSO, superoxide dismutase (SOD), methyl green, Tris(hydroxymethyl)aminomethane, disodium salt of calf thymus DNA (highly polymerized, stored at 4 °C), 4,6-diamino-phenylindole (DAPI), (Sigma-Aldrich), COCl₂ (Fluka), H₂O₂ (Merck) and pBR322 supercoiled plasmid DNA (Genei) were used as received. The ligands L_A and L_B were prepared by the procedure adopted by Carrano et al.48 (link)49 (link).