Goat anti lhx8

All liver and ovary tissues were homogenized with LN2 and lysed on ice with RIPA buffer (SIGMA-Aldrich) containing protease inhibitor cocktail (Roche Diagnostics) and a phosphate inhibitor (A.G Scientific, Inc.). Protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride membranes (PVDF; Bio-Rad). The membrane is blocked with 5% BSA (Amresco) for 1 hour at RT, and then incubated with primary antibodies for overnight at 4° C. Antibodies were used in this study included rabbit anti-NOX4 (1:1000, Abcam), mouse anti-HO-1 (1:1000, Novus) rabbit anti-Catalase (1:1000, Abcam), mouse anti-SOD1 (1:1000, Cell Signaling), rabbit anti-albumin (1:500, Novus), mouse anti-Nobox (1:500, Santa Cruz), rabbit anti-Nanos3 (1:1000, Abcam), goat anti-Lhx8 (1:500, Santa Cruz), rabbit anti-GAPDH (1:1000, Abfrontier) and horseradish peroxidase – (HRP-) conjugated secondary antibody (anti-rabbit IgG; 1:10000; Cell signaling and anti-mouse IgG antibody; 1:5000, Cell Signaling). All experiments were performed in triplicate. Intensity of each band was quantified by Image J software (NIH, Bethesda, http://www.nih.gov).

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).