Rabbit monoclonal anti nanog

Cells were grown in Matrigel coated slide chambers, fixed with freshly prepared 4% paraformaldehyde (PFA) for 15 mins, permeabilized with 0.25% Triton X-100 for 5 mins, blocked with 4% BSA for 1 h, and stained with primary antibody overnight at 4°C. The following primary antibody dilutions were used: rabbit monoclonal anti-Sox2 (1:200, #9092, Cell Signaling Technology), rabbit monoclonal anti-Nanog (1:200, #9092, Cell Signaling Technology), mouse monoclonal anti-α-tubulin (1:200, T6199, Sigma-Aldrich), MF20c (1:50, DSHB), goat polyclonal anti-α-sarcoglycan (1:50, sc-16165, Santa Cruz Biotechnology), and mouse monoclonal anti-β-dystroglycan (1:500, NCL-b-DG, Leica Biosystems, Germany). Secondary antibody dilutions were goat anti-mouse AF488 (1:400, A11029, Thermo Fisher Scientific) and donkey anti-goat AF594 (1:400, A11058, Thermo Fisher Scientific). Finally, cells were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen). Images were obtained with the Invitrogen EVOS FL auto 2 Cell Imaging System. Quantification of α-sarcoglycan expression in differentiated myotubes were done in ImageJ software v2.0 using line intensity plot profile of individual myotubes and normalized with respective DAPI intensity. Percentage of α-sarcoglycan intensity for each group were plotted and statistical analysis was done by Kruskal-Wallis multiple comparison test.

The cells were fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich) for 15 min, incubated with 0.1% Triton X-100/PBS for 15 min, and blocked in 5% BSA/PBS for 1 hour. The cells were then incubated with the following primary antibodies overnight at 4°C: mouse monoclonal anti–TRA-1-60 (1:100, MAB4360; Millipore), mouse monoclonal anti–TRA-1-81 (1:100, MAB4381; Millipore), mouse monoclonal anti–SSEA-4 (1:100, 330402; BioLegend), rabbit monoclonal anti-NANOG (1:1000, 5232; Cell Signaling Technology), rabbit monoclonal anti-OCT4 (1:1000, 5677; Cell Signaling Technology), rabbit monoclonal anti-SOX2 (1:1000, 5024; Cell Signaling Technology), rabbit monoclonal anti-DNMT3B (1:1000, 67259; Cell Signaling Technology), mouse monoclonal anti-FLAG (1:1000, F1804; Sigma-Aldrich), and goat polyclonal anti-GATA6 (1:500, AF1700; R&D Systems). The cells were then washed three times with PBS and incubated with appropriate fluorescently labeled Alexa Fluor secondary antibodies (1:1000; Invitrogen) for 1 hour. The cells were then washed three times with PBS, incubated with 4′,6-diamidino-2-phenylindole (DAPI; 0.5 μg/ml; Molecular Probes) for 10 min, and washed once with PBS. Images were acquired using the Leica DMI6000B inverted fluorescence microscope equipped with the Hamamatsu ORCA-R2 charge-coupled device camera and analyzed with the Leica application suite advanced fluorescence software.