Anti oct3 4 antibody

The number of cells in each blastocyst in all groups was counted by first staining with anti-Oct3/4 antibody (Santa Cruz) to identify inner cell mass (ICM) cells and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Dako, USA) to label nuclei. Five days after IVF, the zona pellucida (ZP) was removed using an acid Tyrode solution (pH 2.5; Sigma-Aldrich). ZP-free blastocysts were fixed in 3.7% paraformaldehyde in 0.02% Triton X-100 in DPBS containing 0.1% BSA (DPBS-BSA) for 30 min at 4°C and permeabilized with 0.1% Triton X-100. Blastocysts were transferred to 5% normal goat serum for 2–4 h at 4°C and then incubated overnight at 4°C with anti-Oct3/4 antibody (Santa Cruz). Blastocysts were then incubated with secondary antibody and co-stained with Alexa Fluor 594 Phalloidin (Molecular Probes, USA) and DAPI. Blastocysts were mounted on a glass-bottomed dish in PBS and imaged using a Zeiss Axiovert 200M fluorescence microscope with Apotome and a 40× oil-immersion objective lens (Carl Zeiss, Germany). Z-stack images (15–20) of individual blastocysts were obtained and analyzed using Axiovision software 4.6 (Carl Zeiss). Oct3/4- and DAPI-positive cells were counted as ICMs; DAPI-only positive cells were counted as the total cell number. The number of trophectoderms (TEs) was calculated as total cell number minus ICM number.

Human iPSCs were cultured on iMatrix-511 coated plate and incubated for 5 to 7 days. After formation of iPSC colonies, they were fixed with 4% paraformaldehyde. Immunofluorescence staining was performed using the following primary antibodies: anti-OCT3/4 antibody (Santa Cruz Biotechnology); anti-SSEA4 antibody (MilliporeSigma). Alexa Fluor 488–conjugated anti-mouse IgG antibody (Jackson ImmunoResearch) was used for the secondary antibody.

Cell surface antigens for BMMSCs and iMSCs were analyzed using flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) as described previously.⁹⁸ (link) Approximately 1.5 × 10⁵ cells were incubated with the following conjugated monoclonal antibodies: TRA-1-60/BUV395 (BD Biosciences), TRA-1-81/Alexa Fluor 674, CD271/fluorescein isothiocyanate (FITC), CD90-phycoerythrin (PE)/Cy7, CD105-allophycocyanin (APC), CD73-BV421, CD11b-PE, CD14-PE, CD19-PE, CD34-PE, CD79a-PE, CD45-PE, CD142-PE, and HLA-DR-PE (BioLegend). Nonspecific fluorescence was determined using isotype-matched mouse monoclonal antibodies (BioLegend). Data were analyzed by collecting 10,000 events on a BD FACSAria using FlowJo 8.8.4 software (BD Biosciences). Intracellular antigens for iPSCs and iMSCs were analyzed using immunohistochemistry. Cells were plated on 24-well plates, fixed with 4% paraformaldehyde, and blocked with 2% FBS-0.2% Triton X-100 in phosphate-buffered saline (PBS) for 1 h at room temperature. After blocking, the cells were incubated with anti-Oct3/4 antibody (1:100, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4°C and then incubated with Alexa Fluor 568-conjugated anti-mouse immunoglobulin G (IgG; Thermo Fisher Scientific) as secondary antibody at room temperature for 1 h. Fluorescent signals were imaged using an inverted microscope (IX83; Olympus, Tokyo, Japan).