Donkey serum

Cells were fixed with 4% paraformaldehyde for 15 min at room temperature, washed with PBS, permeabilised in 0.1% TritonX-100 for 15 min, and blocked with 10% normal donkey serum (Sigma-Aldrich) for one hour. Primary antibodies were diluted in 5% donkey serum and incubated overnight at 4 °C (OCT3/4 (Santa Cruz Biotechnologies, Dallas, TX, USA), Nanog (Abcam, Cambridge, UK), SMA (Abcam), AFP (R&D Systems, Minneapolis, MI, USA), OLIG2 (Millipore), NKX6.2 (Millipore), TUJI (Sigma-Aldrich), Islet 1 (BD bioscience), Hb9 (Developmental studies hybridoma bank), and FUS (Novus, St. Louis, MI, USA)). The next day cells were washed three times with PBS and incubated with anti-donkey Dylight™ secondary antibodies (Thermo Fisher) diluted 1:400 in 5% donkey serum for one hour at room temperature. Nuclei were stained with 1.25 μg/mL Hoechst (Thermo Fisher) for 5 min, washed with PBS three times, and mounted with FluorSave™ Reagent (Merck Millipore).

To immunostain markers of interest in human BM-MSCs, they were osteo-inducted at the density of 1 × 10⁵ cells/well in 48-well plates (BD Biosciences, San Jose, CA). Fourteen days after osteogenic differentiation, induced cells were washed twice with PBS and fixed with PBS (1×) containing 4% paraformaldehyde at RT for 30 min. Cells were permeabilized with 0.1% Triton X-100 in PBS and then incubated overnight at 4 °C in the presence of mouse anti-alkaline phosphatase (ALP; BD Biosciences, San Diego, US) and collagen type I (ColI; Sigma-Aldrich Chemie, Taufkirchen, Germany) primary antibodies diluted 1:200 and 1:1000 in PBS + 0.1% donkey serum (Santa Cruz Biotechnology, Santa Cruz, CA), respectively. The next day, cells were incubated with Alexa Fluor 568-conjugated donkey anti-mouse IgG (H + L) secondary antibody (Life Technologies Europe, Bleiswijk, the Netherlands) diluted 1:400 in PBS + 0.1% donkey serum for 4 h at RT. Finally, the nuclei were stained with Hoechst 33258 staining (Sigma-Aldrich Chemie, Taufkirchen, Germany) for 10 min. Cells were completely washed with PBS after each step. Images were taken with a fluorescence microscope attached to digital color camera. The Mean fluorescence signal intensity was determined using ImageJ (version 4.1; National Institutes of Health, Bethesda, MA).

Expressions of Collagen I, Collagen III, CTGF, Fibronectin, α-SMA, MMP-9, and TIMP-1 proteins were determined with using immunofluorescent staining. To observe hEnSCs or uterine fibrosis and the ratio between MMP-9 and TIMP-1, the slides were washed 3 times with PBS, after which they were embedded in PBS containing 5% donkey serum for 30 min (Santa Cruz Biotechnology, USA). The hEnSCs and uterine sections were incubated with primary antibodies consisting of anti-Collagen I (1 : 100; Proteintech, China), anti-Collagen III (1 : 100; Proteintech, China), anti-CTGF (1 : 100; Proteintech, China), anti-Fibronectin (1 : 200; ABclonal, China), anti-α-SMA (1 : 100; Abcam, UK), anti-MMP-9 (1 : 200; Affinity, China), or anti-TIMP-1 (1 : 200, Affinity, China) at 4°C overnight. On the following day, the hEnSCs or uterine sections were maintained at room temperature for 1 hour and then incubated with secondary antibodies consisting of goat anti-rabbit IgG, Alexa Fluor 549 (Invitrogen, USA), or a mixture of goat anti-rabbit IgG and Alexa Fluor 549 and goat anti-mouse IgG and Alexa Fluor 488 (Invitrogen, USA). The hEnSCs or uterine sections were then incubated with DAPI (Solarbio, China) at room temperature and away from light for 10 min. The staining of hEnSCs or uterine sections was visualized using an inverted fluorescent microscope (Leica, Germany).

The mouse connective tissue-derived fibroblast cell line L-929 was obtained from Shanghai Fu Xiang Biotechnology Co., Ltd. (China). HyClone fetal bovine serum (FBS) was purchased from Shanghai Yu Bo Biological Technology Co., Ltd. (China). LPS (derived from E. coli O111:B4), CCK-8 (cell counting kit-8), 4 % paraformaldehyde, sealing medium and trypsin were purchased from Beyotime Institute of Biotechnology Co., Ltd. (China); MIF was obtained from R & D systems (USA); rabbit anti-mouse TLR4 antibody, FITC-conjugated donkey anti-rabbit IgG secondary antibody and donkey serum were purchased from Santa Cruz Biotechnology, Inc. (USA). Quantitative PCR kit was purchased from Wuhan Google Biotechnology Co,. Ltd. (China). PE-conjugated anti-mouse TLR4/MD2 antibody (Miltenyi Biotec, Inc.) was purchased from Shanghai Univ-bio Co., Ltd. (China).

Differentiated and control bMSC were fixed in a 4% paraformaldehyde (PAF) for 10 min and permeabilized with 0.1% Triton X-100 for 10 minutes. Cells were then rinsed twice in PBS and blocked in donkey serum (Sigma) for 30 min at RT. Cells were incubated over-night at 4°C with primary mouse monoclonal anti AFP or PrP^C antibody (1:50; Santa Cruz Biotechnology and Cayman Chemical, Ann Arbor, MI) or primary goat polyclonal anti NESTIN, MAP2, TRKA antibody (1:50; Santa Cruz Biotechnology) diluted in donkey serum. Then cells were rinsed three times with PBS and incubated with goat anti-mouse or anti-goat IgG conjugated to FITC (1:200 in donkey serum) for 45 minutes. Then cells were again rinsed three times in PBS and mounted under coverslips in a solution containing 4’, 6-diamidino-2-phenylindole (Santa Cruz Biotechnology). Samples were examined under epifluorescence and the results captured by digital photomicroscopy (Olympus, Tokyo, Japan).