Ab150120

After the cultured fibroblasts reached 70–80% confluence, the cells were fixed in 4% formaldehyde for 30 min, then washed with PBS, and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 10 min at room temperature. The cells were treated with 3% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) for 30 min and incubated with primary mouse anti-vimentin antibody (ab8978; Abcam, Cambridge, MA, USA), diluted in PBS (1: 100), at 4°C overnight. After removing the primary antibody, the cells were washed by three times in PBS, and the secondary goat polyclonal anti-mouse IgG antibody was added, which was conjugated with fluorescein isothiocyanate (FITC) (1: 200) (ab150120; Abcam, Cambridge, MA, USA) and incubated at room temperature for 1 h. Then, the samples were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime Biotechnology, Inc., Shanghai, China).

Spermatocyte and oocyte chromosome spreading was prepared as previously described (50 (link), 51 (link)). Structurally preserved spermatocytes were prepared as described previously (52 (link)).
Primary antibodies used for immunofluorescence were as follows: rabbit anti-SYCP3 (1:500 dilution; Abcam #ab15093), mouse anti-SYCP3 (1:500 dilution; Abcam #ab97672), rabbit anti-SYCP1 (C-terminal) (1:2000 dilution; Abcam #ab15090), rabbit anti-SYCP1 (N-terminal) (1:2000 dilution; Abclonal #A12139), rabbit anti-RPA2 (1:200 dilution; Abcam #ab76420), rabbit anti-RAD51 (1:200 dilution; Thermo Fisher Scientific #PA5-27195), rabbit anti-DMC1 (1:100 dilution; Santa Cruz Biotechnology #sc-22768), mouse anti–phospho-histone H2AX (pSer¹³⁹) (1:300 dilution; Millipore #05-636), mouse anti-MLH1 (1:50 dilution; BD Biosciences #550838), rabbit anti-TEX12 (1:1000 dilution; Proteintech #17068-1-AP), mouse anti-TRF1 (1:1000 dilution; homemade), and rabbit anti-BRCA1 (1:500 dilution; a gift from L.-Y. Lu, Zhejiang University). Primary antibodies were detected with Alexa Fluor 488– or 594–conjugated secondary antibodies (1:500 dilution; Abcam #ab150084, #ab150077, #ab150113, and #ab150120) for 1 hour at room temperature. The slides were washed several times with PBS and mounted using VECTASHIELD antifade mounting medium with DAPI (Vector Laboratories, #H-1200).

Eight weeks post transplantation, we estimated the survival rate of hUC-MSCs transplanted into the injured spinal cords. Paraffin sections were dewaxed and rehydrated, and then repaired with antigen. After being blocked, the sections were exposed to primary antibody against human nuclear (HuNu; 1:500, ab216943, Abcam, UK) at 4°C overnight, followed by reaction with secondary antibody goat anti-mouse IgG H&L (Alexa Fluor® 594, ab150120, Abcam, UK). Next, the nuclei were counterstained with DAPI Staining Solution (ab228549, Abcam, UK). The model group functioned as the negative control group. After the sections were mounted with the glycerol jelly mounting medium (S2150, Solarbio, China), the immunofluorescent images were acquired using a fluorescence microscope (BX53, OLYMPUS, Japan).

The hippocampus tissues were separated after MWZ test (Figure 1) and post-fixed at 4°C overnight, followed by the dip in a 30% sucrose solution for 48 h. Sections of 30 µm thick were prepared by a freezing microtome (VT1000S, Leica Microsystems). For all cases, slices were incubated with the mouse anti-doublecortin (DCX, 1:500, ab254133, Abcam), rabbit anti-BrdU (1:200, ab152095, Abcam), mouse anti-neuronal nuclei (NeuN, 1:400, ab104224, Abcam) antibodies. The secondary antibodies were tagged with goat anti-mouse Alexa 594 (1:500, ab150120, Abcam), goat anti-rabbit Alexa 488 (1:500, ab150081, Abcam). Then slices were stained with 4ʹ,6-diamidino-2-phenylindole (DAPI, 0.5µg/mL, 4083s, Cell Signaling Technology). After staining, slices were visualized by two experimenters blinded to group allocation with the laser scanning confocal microscopy (FV1000; Olympus). The mean counts of Brdu/NeuN as well as Brdu/DCX co-labeled cells in every fifth section for each animal was used to evaluate neurogenesis in the DG.