2.6. Immunocytochemistry
Cells were plated on 13 mm round glass coverslips and incubated at 37 ºC in 5% CO2 prior to fixation for immunofluorescence, performing all experiments in triplicate: 1 x 104cells/coverslip plated when cultures were maintained for 24 hours; 2 x 103 cells/coverslip when they were maintained for 5 days; and 2 x 104 cells/coverslip were plated when they were maintained for 7 days. The cells were fixed with 4% paraformaldehyde (PFA: Sigma-Aldrich Chemicals, Spain) in PBS for 20 minutes and after washing in PBS, the cells were incubated for 1 hour at RT in blocking solution: 10% Bovine Serum Albumin (BSA: Sigma, Spain); 0.25% Triton-X 100; and 1% FBS in PBS. The cells were then incubated overnight at 4 ºC with the primary antibodies. Neuronal microtubules were stained with mouse anti-β-III-Tubulin (1:1,000 dilution: ThermoFisher (MA1-118), USA) and anti-GAP43 (1:1,000 dilution: Abcam (ab16053), UK), mesenchymal stem cell progenitors were stained with CD105 (1:100 dilution: Sigma-Aldrich Chemicals (HPA011862), Spain), while neural progenitors were stained with anti-Nestin (1:200 dilution: ThermoFisher (PA5-32517), USA), anti-GFAP (1:3,000 dilution: DAKO (Z0334), USA) and anti-NeuN (1:500 dilution: ThermoFisher (702022), USA), a specific marker for neurons. When anti-Ki67 was used, the cells were permeabilized with 0.2% Triton-X 100 in PBS for 25 minutes and then incubated for 20 minutes in blocking solution containing 4% FBS. The cells were incubated overnight at 4 ºC with anti-Ki67 (1:100 dilution: ThermoFisher, USA) in a buffer containing 0.03% Triton-X 100 and 3% BSA in PBS. The cells were incubated with the secondary antibodies for 1h at RT: AlexaFluor488 donkey anti-Rabbit (1:1000 dilution: Invitrogen (A21206), USA) and AlexaFluor568 donkey anti-Mouse (1:1000 dilution: Invitrogen (A10037), USA). The nuclei were stained with 4′, 6′-diamidino-2-phenylindole dihydrochloride (DAPI, 1:5,000 dilution: Panreac Applichem (A4099), Spain) and all the coverslips were then mounted with Fluoro-Gel medium (Electron Microscopy Sciences, USA). Images were acquired on an MMI CellCut plus (Olympus, Japan).