Three weeks post-infection, cells were trypsinized, seeded on coverslips, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton™ X-100 for 20 min, and blocked for 1 h with PBS containing 0.025% Triton™ X-100, 3% BSA, and 5% FBS. Cells were immunostained overnight at 4°C with the indicated primary antibodies: anti-c-MYC (Cell Signaling Technology), anti-SOX2 (Cell Signaling Technology), anti-NANOG (Cell Signaling Technology), anti-KLF4 (Abcam, UK), anti-OCT3/4 (Cell Signaling Technology), anti-N-cadherin (Cell Signaling Technology), and anti-vimentin (Cell Signaling Technology). After washing with PBS, slides were incubated with appropriate secondary antibodies coupled with Alexa488™ or Cy3 dyes (Molecular Probes, OR, USA). Nuclei were stained with DAPI (Sigma-Aldrich, MO, USA). Coverslips were then mounted with Mowiol® solution (Calbiochem®; Sigma-Aldrich, MO, USA), and the slides were examined with a Zeiss LSM 510 Meta (ZEISS, Germany) confocal microscope. Each microscopy image represents three independent experiments.
Anti oct3 4
Manufactured by Cell Signaling Technology
Sourced in United Kingdom, United States
Anti-OCT3/4 is a primary antibody that specifically recognizes the OCT3/4 (POU5F1) protein. OCT3/4 is a transcription factor that plays a critical role in the self-renewal of undifferentiated embryonic stem cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti nanog, by Cell Signaling Technology (5 mentions)
Anti c myc, by Cell Signaling Technology (2 mentions)
Anti sox2, by Cell Signaling Technology (2 mentions)
Anti vimentin, by Cell Signaling Technology (2 mentions)
Anti n cadherin, by Cell Signaling Technology (1 mentions)
Anti klf4, by Abcam (1 mentions)
Lsm 510 meta, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Anti sox10, by Santa Cruz Biotechnology (1 mentions)
Blocking one, by Santa Cruz Biotechnology (1 mentions)
Alexa 488 or alexa 594 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Slowfade diamond antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Anti gfp, by Thermo Fisher Scientific (1 mentions)
Bz x700, by Keyence (1 mentions)
Anti tra 1 60, by Cell Signaling Technology (1 mentions)
Anti ssea4, by Cell Signaling Technology (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti snail, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Roche (1 mentions)
5 protocols using anti oct3 4
1
Immunofluorescence Staining of Stem Cell Markers
2
Immunocytochemistry of Pluripotent Stem Cells
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Cultured cells were fixed in 4% paraformaldehyde for 10 min at 4°C, permeabilized with 0.2% Triton and 50 mM NH4Cl. Fixed samples were pre-treated with BlockingOne (Nacalai Tesque) for 30 min at RT, and incubated with anti-OCT3/4, anti-NANOG, anti-SSEA4, anti-TRA1-60 (Cell Signaling Technology; diluted 1/200), anti-GFP (Molecular Probes; diluted 1/500), anti-SOX10 (SantaCruz Biotechnology; diluted 1/100) antibodies in 5% of BlockingOne in PBST for overnight at 4°C. After three washes with 0.1% Tween20 in PBS, cells were incubated with Alexa488 or Alexa594-conjugated secondary antibodies (Molecular Probes; diluted 1/500). Cells were washed and mounted in SlowFade Diamond antifade mountant with DAPI (Molecular Probes). Fluorescent images were collected on the software of BZ-X700 (Keyence). For quantitation of cultured cells, numbers of dishes were analyzed from triplicate experiments.
3
Immunoblot Analysis of Epithelial-Mesenchymal Transition
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Cell and tissue samples were lysed in radioimmune precipitation assay buffer (150 mM NaCl, 0.5% sodium deoxycholate, 50 mM Tris-HCl, pH 8, 0.1% SDS, 0.1% Nonidet™ P -40) supplemented with protease inhibitors (Roche AG, Switzerland). Proteins were separated on SDS/PAGE gels, transferred to nitrocellulose membranes, and blocked with 5% nonfat milk in PBS containing 0.1% Tween® 20. The membranes were then probed overnight at 4°C with the relevant primary antibodies: anti-E-cadherin (Cell Signaling Technology, MA, USA), anti-vimentin (Cell Signaling Technology), anti-SNAIL (Thermo Fisher Scientific, MA, USA), anti-ZEB1 (Cell Signaling Technology), anti-OCT3/4 (Cell Signaling Technology), anti-NANOG (Cell Signaling Technology), and anti-GAPDH (Cell Signaling Technology). After extensive washing, the membranes were incubated with the appropriate HRP-conjugated secondary antibodies (Cell Signaling Technology). Blots were detected using an enhanced chemiluminescence detection kit (Amersham BioSciences, UK) and revealed using the ChemiDocTM XRS System (Bio-Rad Laboratories, CA, USA).
4
Immunostaining of Stem Cell Markers
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Cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 95% methanol (MeOH) for 5 min at -20 °C, and washed with PBS containing 0.05% Tween-20. After washing, the cells were blocked with 1% bovine serum albumin (BSA) in PBS for 1 h. The cells were incubated overnight at 4 ℃ with specific primary antibodies: anti-NANOG (1:100, Cell Signaling Technology, Danvers, MA), anti-OCT3/4 (1:100, Cell Signaling Technology, Danvers, MA), anti-Calponin1 (1:500, CNN1; Sigma-aldrich, MO), anti-smooth muscle actin (1:100, SMA; Abcam, Cambridge), anti-sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a, 1:100, Abcam, Cambridge), anti-platelet endothelial cell adhesion molecule (PECAM, Santa Cruz Biotechnology, Dallas, TX), and anti-vascular endothelial cadherin (VECAD, Santa Cruz Biotechnology, Dallas, TX). This was followed by incubation with fluorescent-tagged secondary antibodies (1:100, Invitrogen, Carlsbad, CA). Images were acquired using a confocal laser scanning microscope system (LSM 710, Carl Zeiss AG, Oberkochen) and processed with the Zen software (Carl Zeiss AG, Oberkochen). A water- or oil-immersion objective lens (40 × , or 63 × , 1.4 numerical aperture, NA) with the pinhole set for a section thickness of 0.8 μm (pinhole set to 1 airy unit in each channel) was used.
5
Stem Cell Marker Expression Analysis
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Primer sequences (bp)
Plus SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA) for 60 sec. Protein levels were normalized with respect to the band density of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. The primary antibodies used were: anti-ABCG2 (1:1,000), anti-Bmi-1 (1:1,000), anti-c-Myc (1:1,000), anti-Nanog (1:1,000), anti-Oct3/4 (1:1,000), anti-Sox2 (1:1,000), anti-STAT3 (1:1,000), and anti-ALDH1 (1:1,000) (all from Cell Signaling Technology), and anti-GAPDH (1:5,000) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, uSA).
Statistical analysis. The statistical software SPSS 20.0 (SPSS Inc., Chicago, IL, uSA) was used for data processing and analyzing. Data are presented as the means ± SD, and a comparison was carried between experimental groups of qPCR analysis using one-way ANOVA. The results of the chemosensitivity assay were calculated and compared using one-way ANOVA. P<0.05 was considered statistically significant.
Plus SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA) for 60 sec. Protein levels were normalized with respect to the band density of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. The primary antibodies used were: anti-ABCG2 (1:1,000), anti-Bmi-1 (1:1,000), anti-c-Myc (1:1,000), anti-Nanog (1:1,000), anti-Oct3/4 (1:1,000), anti-Sox2 (1:1,000), anti-STAT3 (1:1,000), and anti-ALDH1 (1:1,000) (all from Cell Signaling Technology), and anti-GAPDH (1:5,000) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, uSA).
Statistical analysis. The statistical software SPSS 20.0 (SPSS Inc., Chicago, IL, uSA) was used for data processing and analyzing. Data are presented as the means ± SD, and a comparison was carried between experimental groups of qPCR analysis using one-way ANOVA. The results of the chemosensitivity assay were calculated and compared using one-way ANOVA. P<0.05 was considered statistically significant.
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