Ab16288

Mature HiNs were fixed overnight in 4% PFA, permeabilized with 1% Triton-X-100 for 5 min at room temperature, and then blocked using 6% normal goat serum at room temperature on a platform rocker for 30 min. HiNs were then incubated in primary antibody (hyperphosphorylated tau: AT8 antibody (ThermoFisher (MN1020) 1:1000), TRA-160 (Abcam (ab16288) 1:200), TRA-181 (Abcam (ab16289) 1:200) overnight at 4 °C on a platform rocker. Neurons were then rinsed 3x in 1xPBS, and incubated in secondary antibody (Alexafluor 594, ThermoFisher 1:1000) at room temperature on a platform rocker for two hours. Fluorescent images were then collected using a 20× air objective using a Nikon Eclipse TE 2000-S microscope (Melville, NY, USA) with an XCite series 120 PC illumination source, captured with Nikon Elements software (AR package, Melville, NY, USA). Data are presented as mean fluorescent intensity in randomly selected ROI in each representative image.

Cells were cultured on a glass slide and fixed in 4% paraformaldehyde for 15 min. The cells were permeabilized with 0.1% Triton X-100 for 10 min, and non-specific antibody interaction sites were then blocked for 15 min. Next, the cells were incubated at 4 °C overnight with primary antibodies, Nanog (ab109250, Abcam, USA), Oct4 (ab200834, Abcam, USA), SSEA4 (ab16287, Abcam, USA), and TRA-1-81 (ab16288, Abcam, USA), to identify ESCs and follicle stimulating hormone receptor (FSHR, 22665-1-AP, Proteintech, China) to identify granulosa cells. On day 2, the cells were incubated with a secondary antibody in a darkroom for 1 h. Finally, the cell nuclei were labeled with Hoechst 33324 for 1 min. After that, the slides were observed and imaged using a fluorescence microscope (DMI 6000, Leica Micro systems, Buffalo Grove, IL, USA).

To determine stem cell characteristics, we performed staining for pluripotent and cell surface markers. Cells were fixed in 4% paraformaldehyde (w/v) and permeabilized in PBS containing 0.2% Triton X-100 for 20 min at room temperature. After subsequent washes in PBS, embryos were blocked for 30 min at room temperature. Slides were incubated separately with TRA-1-60 (1:200, ab16288, Abcam, UK) and Oct4 (1:200, ab27985, Abcam, UK) antibodies overnight at 4 °C, followed by fluorescein isothiocyanate-conjugated goat anti-mouse or anti-rabbit secondary antibodies (1:200) for 1 h at 37 °C and re-washing in PBS. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) at a final concentration of 0.01 mg/ml for 5 min. Immunostained embryos were mounted on glass slides and examined under a confocal laser-scanning microscope (A1-R, NIKON, Japan).

All cells maintained in monolayer cultures were fixed with 4% paraformaldehyde for 10 min at room temperature. The pluripotency of the derived hiPSCs was tested by immunocytochemical staining for OCT4 (Abcam, Cambridge, United Kingdom; ab27985, RRID:AB_776898), NANOG (R and D Systems, AF1997, RRID:AB_355097), TRA-1–60 (Abcam, ab16288, RRID:AB_778563) and TRA-2–49 (Developmental Studies Hybridoma Bank, TRA-2-49/6E, RRID:AB_528071). The secondary antibodies were goat anti-rabbit IgG conjugated with Alexa-488 (Invitrogen, Carlsbad, California; A-11034, RRID:AB_2576217) and goat anti-mouse IgG conjugated with Alexa-488 (Invitrogen, A-11011, RRID:AB_2534069).

Cells were fixed with cold 4% PFA, 4% sucrose in PBS for 10–15 min. For EB2 staining, neurons were fixed with methanol supplemented with 1 mM EGTA for 5 min at −20°C, followed by 5 min of 4% PFA, 4% sucrose based on Jaworski et al Mouse neurons were blocked and permeabilized with 0.2% Triton X‐100/10% normal goat serum in PBS for 60 min at RT. Human cells were treated the same with 0.1% Triton X‐100/3% BSA in PBS. In blocking buffer, the following primary antibodies were incubated o/n at 4°C: mouse anti‐MAP2 (1:1,000, Sigma M9942), rat anti‐EB2 (1:1,000, Abcam ab45767), rabbit anti‐EB2 pS222 (1:1,000), rabbit anti‐MAP1S LC (1:500, Sigma HPA050934), rabbit anti‐OCT4 (1:500, Abcam ab19857) and mouse anti‐TRA‐1‐60 (1:250, Abcam ab16288). After three 5‐min washes with PBS, 1:500 secondary antibodies in PBS were incubated for 2 h at RT and washed three times again with one containing 1:2,000 DAPI stain (Thermo Scientific). Coverslips were mounted with Fluoromount‐G (Southern Biotech) or Prolong Gold Antifade (Invitrogen) on glass slides.