Goat anti nanog

Human cells were fixed in 4% paraformaldehyde in PBS at 4 °C for 10 min, permeabilized at room temperature for 15 min in 0.1% Triton in PBS and then blocked in 5% donkey serum with 0.1% Triton at room temperature for 30 min. The following primary antibodies and dilutions were used: goat anti-NANOG (R&D), 1:200; mouse anti-TRA1-60 (Chemicon), 1:100; mouse anti-human NESTIN (Chemicon), goat anti-SOX2 (Santa Cruz), 1:200; rabbit anti-βIII-tubulin/Tuj 1 (Covance), 1:200; mouse anti-MAP2AB (Sigma), 1:200. Secondary antibodies used include Alexa donkey 488 and 568 anti-rabbit (Life Technologies), Alexa donkey 488 and 568 anti-mouse (Life Technologies), and Alexa donkey 488 and 568 anti-goat (Life Technologies); all were used at 1:300. To visualize nuclei, slides were stained with 0.5 μg ml−1 DAPI (4′,6-diamidino-2-phenylindole) and then mounted with Vectashield.

Coverslips were fixed with 4% paraformaldehyde and immunohistochemistry was performed as previously described (Li et al., 2005 (link)). Antigen-antibody reactions were developed by appropriate fluorescence-conjugated secondary antibodies. Nuclei were visualized by Hoechst staining. Primary antibodies used in this study included mouse anti-Tra-1-60 (1:50; Santa Cruz Biotechnology), goat anti-Nanog (1:500; R&D), mouse anti-SSEA-4 (1:100; Developmental Studies Hybridoma Bank, DSHB), mouse anti-HB9 (1:50; DSHB), rabbit anti-Foxg1 (1:100; Abcam), rabbit anti-Tbr1 (1:1000; Proteintech), mouse anti-βIII-tubulin (Tuj1; 1:100; DSHB) and rabbit anti-Tau (1:200; Sigma-Aldrich). The population of HB9-expressing neurons among total differentiated cells (Hoechst labeled) was counted as described previously (Li et al., 2009 (link)). Briefly, the Zeiss microscope was used to capture images. At least four fields of each coverslip were chosen and counted using ImageJ software (National Institutes of Health). For each group, six coverslips were counted. To quantify axonal swellings, blindly selected fields were imaged from six coverslips per group. The number of axonal swellings was counted (at least 500 neurites were analyzed per group) and divided by the total length of Tau⁺ axons in each field, which were measured using ImageJ software as we described before (Denton et al., 2014 (link)).

iPSC colonies were grown for 3 days on Matrigel™-coated coverslips suitable for immunofluorescence staining. The cells were fixed in 4% paraformaldehyde for 10 min at room temperature, rinsed twice with phosphate-buffered saline (PBS) and permeabilized in 0.5% Triton X-100 diluted in PBS for 10 min. Samples were then blocked with 2% FBS in PBS for 10 min at room temperature. The primary antibodies added were for typical pluripotency markers rabbit anti-Oct3/4 (1:100; Santa Cruz, CA, USA), goat anti-Nanog (1:20; R&D, Minneapolis, MN, USA), mouse anti-Sox2 (1:100; Millipore, Santa Cruz, CA, USA), mouse anti-TRA 1–60 (1:100; Millipore), mouse anti-TRA 1–81 (1:100; Millipore), mouse anti-SSEA4 (1:100; Hybridoma Bank, Iowa City, IA, USA) and kept at room temperature for 1 h. The secondary antibodies were as follows: donkey anti-rabbit Cye 3 (1:100; Chemicon) and donkey anti-mouse/goat Alexafluor 488 (1:100; Invitrogen, Carlsbad, CA, USA). Cells were also stained with DAPI (1:1000; Boehringer, Mannheim, Germany) for nuclei detection, diluted in blocking solution, and added in darkness at room temperature for 1 h. To stain the nucleus, 0.15% DAPI was added. The coverslips with the stained preparations were then mounted on glass. The staining was visualized using laser scanning confocal inverted microscope (Axio Observer.z1, Zeiss, Oberkochen, Germany).