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Anti oct3 4 antibody

Manufactured by Santa Cruz Biotechnology
Sourced in United States

The Anti-Oct3/4 antibody is a laboratory reagent used to detect the expression of the Oct3/4 (Octamer-binding transcription factor 3/4) protein. Oct3/4 is a key transcription factor involved in the regulation of embryonic stem cell pluripotency and self-renewal. This antibody can be used in various techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to analyze the presence and distribution of Oct3/4 in biological samples.

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3 protocols using anti oct3 4 antibody

1

Blastocyst Cell Counting Using Immunofluorescence

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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.
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2

Immunofluorescence Characterization of Human iPSCs

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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.
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3

Phenotypic Characterization of Stem Cells

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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).
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