Mouse anti nanog

Cells were fixed for 15 minutes using 4% paraformaldehyde with 4% sucrose in TBS (TrisHCl pH 7.5, NaCl and MilliQ), washed and permeabilized for 15 minutes with Triton-X100 (0.25%) in TBS. After 30 minutes blocking with donkey serum in TBS-Triton (0.25%), cells were incubated overnight at 4 °C with the following antibodies: rabbit or mouse anti-β3 tubulin, rabbit anti-PAX6 (all Covance), mouse anti-HuCHuD, rabbit anti-OCT4, rabbit anti-GS1 (all Thermo Fisher Scientific), chicken anti-MAP2 (Aves), rabbit anti-Nestin, rabbit anti-TBR1, rat anti-CTIP2, mouse anti-SATB2 (all Abcam), rabbit anti-vGLUT1, mouse anti-GAD65 (both Synaptic Systems), mouse anti-S100B (BD transduction laboratories), mouse anti-NANOG, rabbit anti-OTX2, or mouse anti-GFAP (all Millipore). Subsequently, cells were washed and incubated for 1 hour at room temperature with Alexa secondary antibodies (Thermo Fisher Scientific). DAPI was used to counterstain the nuclei. Images were taken either manually with a Leica DMI 4000B microscope or Zeiss LSM 510 (confocal) or automated with the C7000™ High Content Imaging System (confocal, Yokogawa) or Opera Phenix™ High Content Screening System (confocal, Perkin Elmer).

Cells were fixed for 15’ with 4%PFA/4%sucrose in PBS, washed and permeabilized with Triton-X100 (0.25%) in TBS. For TAU aggregates, 1%Triton-X100 was added to the fixative to remove monomeric proteins. After 30’ blocking, cells were incubated overnight at 4°C with following primary antibodies: mouse anti-β3 tubulin, mouse anti-PAX6 (both Covance), chicken anti-MAP2 (Aves), rabbit anti-Tbr1, rat anti-Ctip2, mouse anti-Nestin (all Abcam), rabbit anti-vGlut2, rabbit anti-vGAT (both Synaptic Systems), mouse anti-OCT4, mouse anti-HuC/D (both Invitrogen), mouse anti-Nanog, mouse anti-RD4 (both Millipore), mouse anti-AT8 (Innogenetics) or AT8 conjugated with Alexa 568. The next day, cells were washed and incubated for 1 hour at RT with Alexa secondary antibodies (Life Technologies). DAPI was used to stain the nuclei. Images were taken with OPERA or CV7000 high content readers or the Leica fluorescence microscope.