Immunostaining of Cell Lineage Markers

IF detection of marker proteins in cells, myotubes, neurospheres or neurosphere-like structures derived from cancer cells was performed exactly as described [11 (link)]. Primary antibodies were Myc (Cell Signaling, #13987. 1:400), Msi1 (Cell Signaling, #5663. 1:200), Sox1 (Abcam, #ab109290. 1:250), Myod1 (Novus Biologicals, #NB100-56511; Cell Signaling, #13812. 1:200), Oct4 (Cell Signaling, #83932. 1:500), Pax3 (Abcam, #ab15717. 1:500), Myoglobin (Cell Signaling, #25919. 1:200), Myosin heavy chain (R&D systems, #MAB4470. 1:200), Map2 (Cell Signaling, #8707. 1:200), Synapsin-1 (Cell Signaling, #5297. 1:200). Secondary antibodies were anti-mouse IgG (FITC-conjugated) (Sigma-Aldrich, #F9137. 1:1,000), Alexa Fluor R 568 donkey anti-rabbit IgG (H+L) (Invitrogen, #A10042. 1:1,000), and Cy3-AffiniPure donkey anti-goat IgG (H+L) (Jackson ImmunoResearch Labs, #705-165-147. 1:1,000), anti-mouse or rabbit Alexa Flour 594 (ThermoFisher Scientific, #A21207, #A21203. 1:500), anti-mouse or rabbit Alexa Fluor 647 (ThermoFisher Scientific, #A31573, #A31571. 1:500). Cell nuclei were counterstained with DAPI. Afterwards slides were rinsed, and coverslips were mounted with anti-fade mounting medium (Invitrogen, #S36936). Cells were viewed with a fluorescence microscope (Zeiss LSM 880).