Rabbit anti lin28

iPSCs were fixed with 4% paraformaldehyde in PBS for 15 min and washed with PBS for 3 times, 5 min each. Cells were then permeabilized and blocked with 0.1% triton X and 2.5% bovine serum albumin in PBS for 1 hour and incubated in primary antibodies overnight at 4 °C (mouse anti-OCT4 from Santa Cruz (cat#SC-5279), 1:250; rabbit anti-LIN28 from Abcam (cat#AB46020), 1:500; mouse anti-SSEA4 from Abcam (cat#MC813), 1:200; rabbit anti-NANOG from GeneTex (cat#GTX100863), 1:300; goat anti-NANOG from R&D (cat#AF1997), 1:250; rabbit anti-H3K27me3 from Millipore (cat#07-449), 1:500); mouse anti-Tra-1-60 from Abcam (cat#AB16288), 1:500; and anti-Tra-1-81 Alexa647 from BD Biosciences (cat#BDB560124), 1:10). After washed with PBS for 3 times, 5 min each, cells were incubated in secondary antibodies if necessary (Alexa Fluor 488 donkey anti-mouse IgG (cat#A21202), 1:500; Alexa Fluor 555 donkey anti-rabbit IgG (cat#A31572), 1:500; Alexa Fluor 488 donkey anti-goat IgG (cat#A11055), 1:500, from Life Technologies) for 1 hour at room temperature and nuclei were stained using DAPI (1:5000).

To analyze the expression and localization of LHX8 and Lin-28 in rat ovaries following the transplantation of PD-MSCs, ovary samples were embedded in OCT compound (Fisher Scientific, Pittsburgh, PA, USA). Five-micron-thick cryostat sections were fixed in cold methanol for 10 min and permeabilized with proteinase K (Dako) for 5 min at RT. The sections were blocked with protein block serum-free buffer (Dako) at RT for 30 min and incubated first with goat anti-LHX8 (1:100 dilution, Santa Cruz) at 4 °C overnight and then with rabbit anti-Lin-28 (1:100 dilution, Abcam) in the dark at RT for 2 h. The mixture of secondary antibodies, including Alexa Fluor 488 chicken anti-goat IgG (1:200 dilution, Invitrogen) and Alexa Fluor 594 goat anti-rabbit IgG (1:200 dilution, Invitrogen), was incubated in antibody diluent (Dako) at RT for 1 h, followed by nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA). The stained coverslips were mounted using a mounting solution to avoid light loss. Images were visualized using an Olympus confocal microscope (× 100 magnification) (Olympus, Tokyo, Japan; https://www.olympus-global.com).