Anti oct4

IPSCs and NPCs grown in chamber slides (ThermoFisher, #177437) were fixed with 4% paraformaldehyde in PBS for 20 min, permeabilized with 0,2% Triton X-100 in PBS and blocked in 6% donkey serum for 1hour. Then, cells were incubated with primary and secondary antibodies. Primary antibodies used were: anti-Nestin (mouse, 1:1000, Stem Cell Technologies, 60091.1); anti-Sox1 (rabbit, 1:1000, Stem Cell Technologies, 60095); anti-Sox2 (rabbit, 1:200, Stemgent, 09–0024); anti-Oct4 (mouse, 1:200, Stem Cell Technologies, 60093.1) and anti-Nanog (rabbit, 1:200, Stemgent, 09–0020). Secondary antibodies (Alexa Fluors, ThermoFisher) were anti-rabbit Alexa 488 (1:200, Invitrogen, A32790) and anti-mouse Alexa 594 (1:200, Invitrogen, A21203). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Roche, 10236276001) and then cover glasses (VWR, ECN631-1575) were mounted on top with Fluoromount-G (Invitrogen, #00-4958-02). Images were acquired using a Zeiss LSM 700 confocal microscope and were processed using Fiji software. Fields based on uniform DAPI staining were selected. 300 cells were analyzed for each NPC marker from all lines.

Cells were fixed with paraformaldehyde (4% in PBS) for 20 min, followed by permeabilization (0.2% Triton X-100 in PBS for 10 min) and blocking (3% BSA in 0.2% Triton X-100 in PBS for 10 min). Human cells were incubated in primary antibody for 1,5 h at room temperature with anti-OCT4 (Stem Cell Technology, #60093, 1:200), anti-PAX6 (Stem Cell Technology, #60094, 1:300) or anti-SOX1 (Stem Cell Technology, #60095, 1:100). Then, cells were washed with PBS and incubated with secondary antibody (Alexa Fluor 488 goat anti-mouse (Thermo Fisher Scientific, A-11029), Alexa Fluor 568 F(ab’)2 Fragment of Goat Anti-Rabbit IgG (H + L) (Life Technologies, #A21069) and DAPI (Life Technologies, #1306) for 45 minutes at room temperature. PBS and distilled water wash were followed before the cover slips were mounted on Mowiol (Sigma, #324590).

Fixed cells with 4% PFA were permeabilized with PBS containing 0.3% Triton X‐100 and then blocked with 3% bovine serum albumin (BSA) (Sigma Aldrich). Human anti‐Nanog (1:1000; rabbit polyclonal, PA1‐097; Thermo Fisher Scientific), anti‐Oct4 (1:400 mouse monoclonal, 60 093; Stem Cell Technologies), anti‐brachyury (1:20 goat polyclonal, AF2085; R&D systems), anti‐Sox17 (1:20 goat polyclonal, AF1924; R&D systems) and anti‐Otx2 (1:20 goat polyclonal, AF1979; R&D systems) primary antibodies diluted in 3% BSA were incubated for 3 hours. The following secondary antibodies: goat anti‐mouse Alexa‐Fluor‐647 (A‐21235; Life Technologies), donkey anti‐goat Alexa Fluor‐594 (A‐11058; Life Technologies) and goat anti‐rabbit Alexa‐Fluor‐488 (A‐11008; Life Technologies) were used for detection. 4',6‐diamidino‐2‐phenylindole (DAPI) was used to counterstain the nuclei. The images were acquired with Leica DMi8 inverted microscope, filter cubes and software from Leica microsystems.