Ab109250

IHC analysis was performed on a tissue microarray (TMA) of tumor samples of serous ovarian carcinoma. After evaluation of HE stained sections of the ovarian tumor, the biopsy of representative areas of the tumor tissue was performed with a 2 mm core needle. The biopsy specimens were then transferred to multitumor paraffin blocks. 3-5 μm sections were consecutively cut from the multitumor block. The 3-5 μm thick paraffin sections were then placed on silane-coated slides (Menzel-Glaser Superfrost) and were dried for one hour in a dryer at 60 °C. IHC staining was performed by an automatic slide stainer (Ventana BenchMark GX). After deparaffinization, the antigen retrieval was performed. Antigen retrieval (HIER) was performed with a CC1 Ventana reagent at pH 7-8 for 48 min. The primary antibody was then applied. Rabbit anti-human NANOG monoclonal antibody (ab109250, Abcam Cambridge, MA, USA) was used as the primary antibody at a dilution rate of 1:25. Incubation with NANOG antibody lasted for 30 min at 37 °C. The antigen detection was performed with the Ventana OptiView Kit. The removal of the primary antibody served as a negative control. The stained slides were observed under microscope. The positive reaction of the NANOG protein was visible as yellow-brown staining of the nuclei of cells of the ovarian serous carcinoma.

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).

For fluorescent microscopy, the cultured cells were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilised with 0.1% Triton X-100 for 10 min. After blocking for 30 min with 5% bovine serum albumin, the washed cells were incubated for 1 h at room temperature or overnight at 4°C with a primary antibody against the POU domain, class 5, transcription factor 1 (OCT4) (1:200, ab19857, Abcam), Transcription factor SOX-2 (SOX2) (1:200, ab92494, Abcam), Homeobox protein NANOG (NANOG) (1:100, ab109250, Abcam), Tra-1-60 (1:150, MAB4360, Merck), Tubulin β-3 (TUBB3) (1:500, ab18207, Abcam), smooth muscle alpha actin (SMA) (1:400, A2547, Merck) or α-fetoprotein (AFP) (1:200, ab3980, Abcam). Subsequently, the cells were incubated with fluorescence-labelled secondary Alexa Fluor 594 or 488 (1:1000, A11001, A11008 and A11012, Invitrogen) at room temperature for 1 h, protected from light. The cells were further incubated with 1 µM DAPI for nuclear staining. Between incubations, samples were washed with PBS containing 0.1% Triton X-100. Alkaline Phosphatase Live Stain 500X from Thermo Fisher Scientific was used for determining the enzyme activity following the manufacturer's instructions. Pictures were acquired using a Floyd Cell imaging station (Life Technologies).

Western blot was carried out as previously reported (Wang et al., 2012 (link)). The anti-ING4 (ab108621, 1:1,000; Abcam, United States), anti-CD44 (ab189524, 1:1,000, Abcam, United States), anti-OCT4 (ab200834, 1:1,000, Abcam, United States), anti-MYC (ab32072, 1:1,000, Abcam, United States), anti-NANOG (ab109250, 1:1,000, Abcam, United States), anti-DUSP4 (ab216576, 1:1,000, Abcam, United States), anti-p-p38 MAPK (#4511, 1:1,000, CST, United States), anti-p38 MAPK (#8690, 1:1,000, CST, United States), anti-p-Erk1/2 (#4370, 1:1,000, CST, United States), and anti-Erk1/2 (#4695, 1:1,000, CST, United States) were used for primary antibody incubation at 4°C overnight. The anti-α-tubulin (Cat No.: 11224-1-AP, 1:1,000; Proteintech, China) was used for the protein loading control. Each blot was repeated three times.

Western blot was conducted as previously described [19 (link)]. The proteins were extracted from the breast cancer specimens using RIPA lysis buffer (Beyotime, Shanghai, China). BCA Protein Assay Kit was used to evaluate the protein concentration. The protein was separated from the sample buffer using SDS-PAGE, transferred into PVDF membranes and blocked with 5% skim milk for 1 h. The primary antibodies were provided by Abcam Company, including anti-Nanog (ab109250, 1:1000), anti-Oct4 (ab184665, 1:1000), anti-SOX2 (ab137385, 1:1000). The member was incubated at 4 °C overnight and then incubated with horseradish peroxidase-conjugated secondary antibodies. Immunoblots were visualized by ECL detection system (Pierce, Rockford, IL, USA).