Immunocytochemistry was performed as described before 34 (link) using anti-OCT4 (sc-5279, Santa Cruz Biotechnology) and anti-TROP2 (BD Bioscience) antibodies. Cell nuclei were counterstained with DAPI.
Anti oct4 sc 5279
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-OCT4 (sc-5279) is a primary antibody produced by Santa Cruz Biotechnology. It is designed to detect the OCT4 protein, which is a transcription factor involved in the regulation of embryonic stem cell pluripotency.
Automatically generated - may contain errors
Lab products found in correlation
Ab80892, by Abcam (2 mentions)
Anti nanog sc 293121, by Santa Cruz Biotechnology (2 mentions)
Anti sox2 mab2018, by R&D Systems (2 mentions)
Anti flag tag, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Anti trop2, by BD (1 mentions)
Complete protease inhibitor, by Merck Group (1 mentions)
Anti sox2 sc 17320, by Santa Cruz Biotechnology (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
Pepli g wta single cell kit, by Qiagen (1 mentions)
Da1016, by Solarbio (1 mentions)
P6148, by Merck Group (1 mentions)
Anti map2 sc 20172, by Santa Cruz Biotechnology (1 mentions)
Fluoromount aqueous mounting medium, by Thermo Fisher Scientific (1 mentions)
Ix51 inverted light microscope, by Olympus (1 mentions)
Anti fmrp blg 834601, by BioLegend (1 mentions)
A11012, by Thermo Fisher Scientific (1 mentions)
D1306, by Thermo Fisher Scientific (1 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (1 mentions)
15 protocols using anti oct4 sc 5279
1
Immunocytochemical Detection of OCT4 and TROP2
2
Fetal Gonad Immunohistochemistry and Immunofluorescence
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Human fetal gonads were isolated from material available following elective surgical termination of pregnancy during the first trimester at the Department of Gynecology at Copenhagen University Hospital (Rigshospitalet) and Hvidovre Hospital, Denmark. The regional ethics committee approved this study (permit number H-1-2012-007) and women gave their informed written and oral consent. None of the terminations were for reasons of fetal abnormality and all fetuses appeared morphologically normal. The fetuses in this collection were between gestational week (GW) 7 and 12, with fetal age determined by scanning crown–rump length. Fetal testis tissue was isolated in ice-cold phosphate buffered saline (PBS) and immediately fixed in formalin. Immunohistochemistry was conducted as previously described with anti-DHX37 (HPA047607, Sigma/Prestige Antibodies) diluted 1:50 and antigen retrieval in TEG buffer (10 mM Tris, 0.5 mM EGTA, pH 9.0).24 (link) Immunofluorescence was conducted as previously described,25 (link) with anti-DHX37 (HPA047607, Sigma/Prestige Antibodies) diluted 1:200 and anti-OCT4 (sc-5279, Santa Cruz Biotechnology) diluted 1:100. Immunofluorescence on the human cell lines RT4, KGN, and HEK 293 was performed as described elsewhere10 (link) using anti-DHX37 (HPA047607, Sigma/Prestige Antibodies) diluted 1:200.
3
Pluripotency Marker Profiling via Western Blot
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Cells were harvested in 150 μL of lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40) supplemented with complete protease inhibitors (Sigma) and phosphatase inhibitors (Roche). Cell lysates were separated on 10% SDS-PAGE and blotted onto an NC membrane. The membrane was then blocked in 5% nonfat milk and incubated with primary antibodies. Anti-Phos-Ser/Thr (NC9080, CST), anti-OCT4 (sc-5279, Santa Cruz), anti-SOX2 (sc-17320, Santa Cruz), anti-SOX17 (24,903-1-AP, Proteintech), anti-NANOG (ab80892, Abcam), and anti-GATA6 (55,435-1-AP, Proteintech) antibodies were used for immunoblotting. HRP-conjugated anti-mouse (7074S, CST), anti-rabbit (7076S, CST), or anti-goat (7073S, CST) antibodies were used as the secondary antibody. The images were visualized using the Bio-Rad ChemiDoc XRS+ system.
4
Knockdown of Pluripotency Markers in Mouse Zygotes
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The collection of zygotes was performed as previously described [18 (link)]. CD1 female mice were super-ovulated with 5 IU PMSG (367222, Calbiochem) and 5 IU hCG (230734, Calbiochem) for 46 h and used for breeding. After release from the oviduct ampullae, the zygotes were injected with siRNA oligos (20 μM) using the Femojet microinjection system (Eppendorf) and then cultured in KSOM medium for different amounts of time.
After culturing the zygotes for 24 h (two-cell stage), whole transcriptome amplification was performed using the PEPLI-g WTA single cell kit (Qiagen, 150063). Following cDNA synthesis and amplification, the product was used for qRT-PCR to access the knockdown efficiency and the expression of pluripotency markers.
For whole-mount staining, blastocysts were collected into embryo GPS dishes (LifeGlobal Group), fixed with 4% paraformaldehyde, permeabilized in 0.1% Triton X-100, blocked with 3% BSA solution, and blotted with anti-Oct4 (sc-5279; Santa Cruz) and anti-Nanog (Ab80892; Abcam) antibodies.
After culturing the zygotes for 24 h (two-cell stage), whole transcriptome amplification was performed using the PEPLI-g WTA single cell kit (Qiagen, 150063). Following cDNA synthesis and amplification, the product was used for qRT-PCR to access the knockdown efficiency and the expression of pluripotency markers.
For whole-mount staining, blastocysts were collected into embryo GPS dishes (LifeGlobal Group), fixed with 4% paraformaldehyde, permeabilized in 0.1% Triton X-100, blocked with 3% BSA solution, and blotted with anti-Oct4 (sc-5279; Santa Cruz) and anti-Nanog (Ab80892; Abcam) antibodies.
5
Immunohistochemical Analysis of Stem Cell Markers
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Paraffin-embedded tissue samples were cut into 5-μm thick sections, deparaffinized with xylene, and dehydrated using graded alcohol washes. Antigen retrieval was performed for all sections by heating in a microwave oven, and endogenous peroxidase activity was blocked with 3% H2O2 solution. After 1 h of serum blocking, anti-NANOG (sc-293121, Santa Cruz) or anti-OCT4 (sc-5279, Santa Cruz) antibodies were added to samples and incubated overnight at 4 ℃. The samples were then incubated with a secondary antibody, and then a chromogenic agent (DA1016, Solarbio) was added. In addition, human HCC tissues were stained with Sirius Red. Immunohistochemistry results were analyzed using ImageJ software, and the final results were presented using the average optical density.
6
Immunocytochemical Analysis of Pluripotency Markers
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To perform immunocytochemistry analysis, the cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 15 min at room temperature. Fixed cells were washed twice with ice-cold PBS and were subsequently incubated with PBS containing 0.1% Triton X-100 for 10 min, and washed again in PBS for three times. After blocking unspecific bindings with BSA-blotting buffer (1% BSA, 0.1% Tween 20 in PBS) for 30 min, the cells were incubated with BSA-blotting buffer and antibodies of anti-SSEA1 (4746s, Cell Signaling Technology, USA), anti-Tra-1–60 (4746s, Cell Signaling Technology), anti-OCT4 (SC-5279, Santa Cruz Biotechnology, USA), anti-NANOG (ab80892, Abcam, USA), and anti-SOX2 (3579s, Cell Signaling Technology) kept in a humidified chamber at 4 °C overnight or at 37 °C for 1 h. After washing with PBS for three times, the cells were incubated with FITC conjugated secondary antibody for 1 h at room temperature. The nuclei were stained by 10 µg/mL Hoechst 33342 for 2 min. The images were documented by an EVOS fluorescence microscope.
7
Immunocytochemical Characterization of Cells
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Cells were fixed with 4% paraformaldehyde (PFA; P6148, Sigma-Aldrich) for 20 min at room temperature (RT). Blocking was performed with blocking solution: 10% Fetal Bovine Serum (FBS) or 5% Goat serum (GS) with 0.2% Triton X 100 in PBS for permeabilization. Cells were then incubated with primary antibodies (anti-SSEA4, CST-4755, Cell Signaling Technology, Danvers, MA, USA; anti-TRA-1-60, ab16288, Abcam, Cambridge, UK; anti-OCT4, sc-5279, Santa-Cruz, Starr County, TX, USA; anti-FMRP, BLG-834601, Biolegend, San Diego, CA, USA; anti-Tuj1, BLG-801201, Biolegend; anti-PAX6, BLG-901301, Biolegend; anti-MAP2, sc-20172, Santa Cruz; anti-SYN1, AB1543, Merck, Darmstadt, Germany; anti-PSD-95, MAB1596, Merck) diluted in blocking solution for 1 h at RT, washed 3 times with PBS, and incubated with secondary antibodies (donkey anti-mouse Alexa Fluor 488, A21202, Thermo Fisher Scientific, Waltham, MA, USA; goat anti-rabbit Alexa Fluor 594, A11012, Thermo Fisher) for another 1 h at RT in the dark, and counterstained with DAPI for nucleus localization (D1306, Thermo Fisher Scientific). Cells were mounted with Fluoromount aqueous mounting medium (00-4958-02, Thermo Fisher Scientific). Bright-field, phase, and fluorescence images of cells were obtained using an Olympus IX51 inverted light microscope (Olympus, Tokyo, Japan).
8
qRT-PCR and Imaging of Mouse Gonads
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For quantitative reverse-transcription PCR (qRT-PCR) of mouse tissue, messenger RNA (mRNA) was extracted from subdissected gonads of 6 XX and 6 XY 11.5 dpc mouse embryos using the RNeasy Plus Micro Kit (Qiagen). Each sample was then mixed with FastSYBR green master mix (Applied Biosystems) and either Dhx37 or Hrpt1 primers, plated in duplicate, and run on a 7500 Fast Real-Time PCR System (Applied Biosystems). Whole-mount in situ hybridization of 11.5 dpc wild-type (WT) XY gonads has been previously described.26 (link) Immunofluorescence staining with anti-DHX37 (HPA047607, Sigma/Prestige Antibodies), anti-OCT4 (SC-5279, Santa Cruz Biotechnology), and DAPI (Vectashield with DAPI, Vector Laboratories) was performed on transverse sections of 11.5 dpc XY gonads. Sections were imaged at 20× using an LSM 700 Inverted Confocal Microscope (Zeiss).
9
Western Blot Protein Analysis
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Western blotting was performed as described previously [14 (link)]. We used the following primary antibodies: anti-Orai3 (ab115558, Abcam, Cambridge, UK), anti-ID1 (sc-133104, Santa Cruz, CA, USA), anti-ID2 (sc-398104, Santa Cruz, CA, USA), anti-Bmi1 (5856, Cell Signaling), anti-EZH2 (ab186006, Abcam, Cambridge, UK), anti-Nanog (sc-293121, Santa Cruz, CA, USA), anti-Oct4 (sc-5279, Santa Cruz, CA, USA), and anti-GAPDH (sc-25778; Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Santa Cruz.
10
Western Blot Analysis of Protein Expression
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Western blot analysis was performed as described previously [47 (link)]. Anti-ACTB (A2228) antibody was purchased from Sigma–Aldrich; anti-Flag (#2368) was from Cell Signaling Technology (Danvers, MA, USA); anti-OCT4 (sc-5279) and anti-PP1γ (sc-6108) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-p-OCT4 (S236) antibody detecting human p-OCT4 (S236) [corresponding to murine p-Oct4(S229)] was identical to the anti-p-Oct4(S229) antibody described previously [34 (link),35 (link)]. Goat anti-mouse IgG (#31430) was purchased from Pierce (Waltham, MA, USA). Goat anti-rabbit IgG (#1706515) and rabbit anti-goat IgG (H+L)-HRP conjugate (#1721034) were purchased from Bio-Rad (Hercules, CA, USA).
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