Journal: International Journal of Molecular Sciences

Article Title: Direct On-Chip Differentiation of Intestinal Tubules from Induced Pluripotent Stem Cells

doi: 10.3390/ijms21144964

Figure Lengend Snippet: Characterization of hiPSC-derived human intestinal-like tubules. ( A ) 3D reconstruction image of an iPSC-derived tubule at Day 4 stained with antibodies for SOX17 (green), FOXA2 (red) or DAPI for DNA (blue). Cells attach and form tubule at the DE stage. ( B ) Representative images of iPSC tubules stained for Definitive endoderm markers SOX17 and FOXA2 at day 4. ( C ) Hindgut marker CDX2 at day 7. ( D ) Gene expression was measured using TaqMan qRT-PCR from hiPSC tubules at different differentiation stages. The following genes were analysed: POU class 5 homeobox 1 (POU5F1, indicating pluripotency); Nanog homeobox (NANOG, indicating Primitive Streak):, forkhead box a2 (FOXA2, Definitive Endoderm), SRY (sex determining region Y)-box 17 (SOX17, Definitive Endoderm), and Homeobox protein CDX-2 (Posterior gut). ( E ) Representative images of iPSC tubules stained for intestinal markers Lysozyme (LYZ), Villin (VIL) and Chromogranin A (CHGA) at day 28 (green). Nuclei were stained with DAPI (blue) to visualize the overall morphology. Scale bars = 100 µm. ( F ) Relative gene expression analysis of Intestinal markers: Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), Mucin-2 (MUC2), Lysozyme (LYZ), Villin-1 (VIL1), Chromagranin A (CHGA) and sucrase-isomaltase (SI). ( G ) Relative mRNA expression of MDR1 (P-gp) and CYP3A4 in iPSC-derived intestine-like tubules on day 14, day 28 and day 31, human adult colon organoids and Caco-2 cells. Expression levels were normalized to beta-actin (ACTB), data represented as mean ± SD relative to the expression in undifferentiated miFF1 hiPSC ( n = 2, n ≥ 2–3). The Y-axis represents the LOG10 relative quantification (RQ). Caco-2 cells and primary colon organoid were also included to compare gene expression and to follow the differentiation of our model during the different stages. Data are presented as the average of two independent experiments +/− SD ( n = 3).

Article Snippet: 1/100) CDX2 (R&D Systems, AF3665, 1/100), Villin (Santa Cruz, 58897, 1/200), Chromagranin A (Rabbit Santa, cruz13090, 1/200), Lysozyme (Thermo Fischer, MA1-82873,1/200), Isotype Rabbit (Life Technologies, 86199, 1/250) Isotype Goat (Life Technologies, 026202, 1/250), Isotype Mouse (Invitrogen, 08-6599 1/250), Goat Alexa Fluor 488 (Invitrogen, A11055 1/250) Mouse Alexa Fluor 555(Life Technologies, 1736967, 1/250), Rabbit Alexa Fluor 647 (MERCK, SAB460177 1/250) and cell stains Actin Green (LifeTechnologies, R37110), Hoechst 33342 (Thermo Fischer, H3570, 1/1000).

Techniques: Derivative Assay, Staining, Marker, Gene Expression, Quantitative RT-PCR, Expressing, Quantitative Proteomics

Journal: Diagnostic Pathology

Article Title: Colon adenoma and adenocarcinoma with clear cell components - two case reports

doi: 10.1186/s13000-019-0819-z

Figure Lengend Snippet: Tubular adenoma with clear cell change. The striking tubule structures of the clear cells are accompanied by conventional tubular adenoma cells at low magnification with HE staining ( a ). The boundary between the clear cell and conventional components at high magnification with HE staining ( b ). The clear cell component is negative for PAS ( c ) and alcian blue staining ( d ). Both components are positive for CDX2 staining ( e ). The localization of CEA (f) expression is diffusely cytoplasmic for the clear cell component, and luminal cell apical for the conventional one. Ki67 labeling ( g ) is slightly lower in the clear cell component. d-g represent immunohistochemistry. Ultrastructural examination ( h ) of the boundary between the clear cell area (left) and the conventional adenoma (right) at low magnification is shown and multiple cytoplasmic lipid-like vacuoles surround the nuclei in the clear cells ( i )

Article Snippet: CDX2 , AMT28 , Novus Biologicals, Newcastle, UK , 1:50 , pH 9.0.

Techniques: Staining, Expressing, Labeling, Immunohistochemistry

Journal: Diagnostic Pathology

Article Title: Colon adenoma and adenocarcinoma with clear cell components - two case reports

doi: 10.1186/s13000-019-0819-z

Figure Lengend Snippet: Clear cell adenocarcinoma. Low magnification ( a ) and high magnification of the clear cell ( b ) and conventional components ( c ) with HE staining. The boundary ( d ) between the clear cell and conventional components at high magnification with HE staining. The clear cells are negative for PAS ( e ) and alcian blue staining ( f ), whereas both components of the tumor are positive for CDX2 ( g ). The localization of CEA ( h ) expression is diffusely cytoplasmic for the clear cell component and luminal cell apical for the conventional component. CD10 ( i ) and adipophilin ( j ) expression is confined to the clear component and Ki67 labeling ( k ) is higher in the clear cell component. g-k represent immunohistochemistry. Immunoelectron microscopy analysis ( l ) at low (left) and high magnification (right) reveals multiple cytoplasmic lipid-like vacuoles in clear cells that are negative for adipophilin

Article Snippet: CDX2 , AMT28 , Novus Biologicals, Newcastle, UK , 1:50 , pH 9.0.

Techniques: Staining, Expressing, Labeling, Immunohistochemistry, Immuno-Electron Microscopy

Journal: Diagnostic Pathology

Article Title: Colon adenoma and adenocarcinoma with clear cell components - two case reports

doi: 10.1186/s13000-019-0819-z

Figure Lengend Snippet: Antibody information

Article Snippet: CDX2 , AMT28 , Novus Biologicals, Newcastle, UK , 1:50 , pH 9.0.

Techniques:

Journal: PLoS ONE

Article Title: Brg1 Is Required for Cdx2-Mediated Repression of Oct4 Expression in Mouse Blastocysts

doi: 10.1371/journal.pone.0010622

Figure Lengend Snippet: (A) qRT-PCR analysis of Brg1, Cdx2, and Oct4 transcripts in Brg1 KD 8-cell embryos, morulae, and blastocysts. Data were normalized to Ubtf (house keeping gene) and are relative to control embryos at each stage; black line = 1. Asterisk denotes significant difference between Brg1 KD and control blastocysts (p<0.05). (B) ICC analysis of Oct4 and Cdx2 expression in Brg1 KD 8-cell embryos, morulae, and blastocysts. Nuclei were counter stained with DAPI (blue). (C) Brg1 represses Oct4 expression in a dose dependent manner. One-cell embryos were injected with 0 µM (control), 0.1 µM, 1 µM, or 100 µM Brg1 siRNA and cultured to the blastocyst stage. Real-time qPCR was used to analyze the levels of Brg1 and Oct4 transcripts. Data were normalized to Ubtf and are relative to control blastocysts; dashed line = 1.

Article Snippet: CDX2 was detected by Western blot analysis using an affinity purified rabbit anti-CDX2 antibody (A300-692A, Bethyl Laboratories, Inc).

Techniques: Quantitative RT-PCR, Control, Expressing, Staining, Injection, Cell Culture

Journal: PLoS ONE

Article Title: Brg1 Is Required for Cdx2-Mediated Repression of Oct4 Expression in Mouse Blastocysts

doi: 10.1371/journal.pone.0010622

Figure Lengend Snippet: (A) ICC analysis of Oct4 and Cdx2 in Brg1 KD and control blastocysts. In control blastocysts (a–d) Oct4 expression (green) is restricted to the ICM and is largely absent in the Cdx2-positive (red) trophectoderm. In contrast, in Brg1 KD blastocysts (e–h) Oct4 is widely expressed in both the ICM and cdx2-positive (yellow) trophectoderm. Arrowheads denote co-expression of Oct4 and Cdx2. Nuclei were counter stained with DAPI (blue). (B) Quantification of the average number of cells in Brg1 KD and control blastocysts expressing Oct4, Cdx2, and Oct4 & Cdx2 (double expression). Asterisks denote statistical significance (p<0.05) between Brg1 KD and control blastocysts. A total of 25 Brg1 KD blastocysts and 15 control blastocysts were analyzed.

Article Snippet: CDX2 was detected by Western blot analysis using an affinity purified rabbit anti-CDX2 antibody (A300-692A, Bethyl Laboratories, Inc).

Techniques: Control, Expressing, Staining

Journal: PLoS ONE

Article Title: Brg1 Is Required for Cdx2-Mediated Repression of Oct4 Expression in Mouse Blastocysts

doi: 10.1371/journal.pone.0010622

Figure Lengend Snippet: (A) Combined depletion of Brg1 and Cdx2 augments Oct4 expression in blastocysts. qRT-PCR analysis of Oct4 transcripts in Brg1 KD blastocysts, Cdx2 KD blastocysts, and Brg1 & Cdx2 double KD blastocysts. Data were normalized to Ubtf (house keeping gene) and are relative to control blastocysts; dashed line = 1. Different letters denote statistical significance in Oct4 transcripts (p<0.05). These experiments were replicated using a total of 5 biological replicates. (B) Co-immunoprecipitation and western blot analysis of Brg1 and Cdx2 in TS cells. Brg1 was immunoprecipitated using a rabbit anti-serum. Recovery of Cdx2 was measured by western blot analysis. Cdx2 is enriched in the Brg1 IP samples and not in the control IgG samples. This assay was repeated a total of 4 times using different batches of TS cells. (C) Confocal immunofluorescence analysis of Brg1 and Cdx2 in blastocysts. Co-localization of endogenous Brg1 and Cdx2 in trophectoderm nuclei was determined using specific antibodies for Brg1 and Cdx2. Nuclei were counterstained with DAPI. White box represents magnified region in bottom panel. Arrow denotes blastocyst ICM. (D) Confirmation of Flag-Cdx2 expression in induced ES cells. Western blot and immunofluorescence analysis of Flag-Cdx2 expression at 24 hours following removal of doxycycline. (E) ChIP analysis of Brg1 and Cdx2 binding to the Oct4 promoter in Cdx2-inducible ES cells. qRT-PCR was used to determine the relative enrichment of Brg1 and Flag-Cdx2 at the Oct4 ARE versus an intergenic region in uninduced and induced ES cell extracts. A non-specific rabbit IgG was included as a negative control. Data were normalized to 1% input DNA. Asterisks denote significant differences between uninduced and induced samples (p<0.05). These experiments were replicated 3 to 4 times using two different batches of Cdx2-inducible ES cell extracts.

Article Snippet: CDX2 was detected by Western blot analysis using an affinity purified rabbit anti-CDX2 antibody (A300-692A, Bethyl Laboratories, Inc).

Techniques: Expressing, Quantitative RT-PCR, Control, Immunoprecipitation, Western Blot, Immunofluorescence, Binding Assay, Negative Control

Journal: PLoS ONE

Article Title: Brg1 Is Required for Cdx2-Mediated Repression of Oct4 Expression in Mouse Blastocysts

doi: 10.1371/journal.pone.0010622

Figure Lengend Snippet: Schematic diagram of Oct4 regulation in blastocysts. In the trophectoderm Brg1 is recruited to the ARE of the Oct4 promoter via Cdx2. Once at the Oct4 promoter Brg1 and Cdx2 facilitate recruitment of additional co-repressors to repress transcription.

Article Snippet: CDX2 was detected by Western blot analysis using an affinity purified rabbit anti-CDX2 antibody (A300-692A, Bethyl Laboratories, Inc).

Techniques:

Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 1. Genomic organization of the human ELF5 locus and transcript isoform expression in placenta and trophoblast cell lines. (A) Diagram of the exon–intron structure of the human ELF5 locus and annotated splice var- iants. Position of primers used is indicated. Filled boxes represent open- reading frames and open boxes represent untranslated regions. (B) RT–PCR analysis with isoform-specific and common primers reveals that ELF5-2b is the expressed splice variant in placenta and the trophoblast-like cell line TCL-1, but that it is absent from the first trimester mesenchymal-like cell line TCL-2. (C) RT–PCR with primers spanning exons 3 and 4 demonstrates that the annotated ELF5-2bDex3/4 variant is not present in placenta and chor- iocarcinoma and trophoblast-like cell lines JEG-3 and TCL-1.

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Variant Assay

Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 2. Expression of trophoblast stem cell genes and epigenetic regulation of ELF5 in placenta throughout gestation. (A) RT–PCR analysis of ELF5, CDX2 and EOMES (i.e. genes important for trophoblast stem cell self-renewal and proliferation in the mouse) on human placental villous samples ranging from 7 weeks of gestation to term. Four independent term placental samples were investigated. The choriocarcinoma cell line JEG-3 was included as control. Colour-inverted photographs of ethidium bromide stained gels are shown. All three genes are expressed in placenta, but CDX2 is not detected from the second trimester onwards even when the PCRs are over-cycled. (B) Quantitative RT–PCR (qPCR) analysis of ELF5, CDX2 and EOMES on the same samples used in (A). ELF5 is down- regulated in second and third trimesters, whereas no overall regulation with gestational age was observed for EOMES. (C) Comparison of expression levels between first trimester and term. ELF5 expression is significantly reduced at term when compared with first trimester, CDX2 is absent from term placentas. (D) Bisulphite sequencing analysis of the ELF5 promoter region. Filled circles indicate methylated cytosine residues. ELF5 is extremely hypomethylated in the first trimester and acquires higher DNA methylation levels in second and third trimester, correlating with transcriptional down-regulation at these stages. (E) DNA methylation analysis of an extended region between 2400 bp and +400 bp around the transcriptional start site of ELF5. Hypomethylation correlates with ELF5 expression in JEG-3 cells and, conversely, ELF5 is hypermethylated and not expressed in TCL-2 cells. The methylation pattern in TCL-1 cells reveals a critical stretch of five CpG residues (grey box) at the immediate transcriptional start site that needs to be unmethylated for ELF5 to be expressed.

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Staining, Quantitative RT-PCR, Comparison, Bisulfite Sequencing, Methylation, DNA Methylation Assay

Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 3. Immunofluorescence localization of ELF5 to cytotrophoblasts in the human placenta. (A) Overview of 11 week placental villous cross-section shows ELF5 localization to nuclei of villous cytotrophoblasts, but absence from nuclei of the overlying syncytiotrophoblast layer. Cytotrophoblasts are a proliferative cell population that continuously divide to replenish the overlying syncytium. (B) Co-localization with cytokeratin 7 (CK7) confirms the trophoblast identity of ELF5-positive cells. (C) Confocal image of a double staining of ELF5 and the villous cytotrophoblast marker SPINT1 (also known as HAI-1) shows that every ELF5-positive nucleus resides within the cytotrophoblast layer. Top row 6 week, bottom row 11 week placenta. (D) Confocal image analysis of an 11 week villous section stained for ELF5 and the extravillous cytotrophoblast (EVT) marker integrin alpha-5 (ITGA5). ELF5 is detected only in nuclei at the proliferative base, but not further distal along the EVT column where cells adopt an invasive phenotype and lose proliferative potential. (E) ELF5 is also absent from post- mitotic interstitial and endovascular EVTs within the decidual bed.

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Double Staining, Marker, Staining

Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 4. CDX2 identifies a subset of ELF5-positive cytotrophoblasts as a TS-like compartment that is regulated by FGFR2. (A) ELF5 co-localizes with FGFR2 in villous cytotrophoblasts as identified by confocal image analysis of double immunofluorescence stainings of 11 week placental sections. Since FGF signalling has been implicated in TS cell proliferation in mice and humans and can activate ELF5 expression in other tissues, FGF/FGFR2 may induce ELF5 expression within a putative TS cell niche in the human placenta. (B) Double staining of a 6 week placental section for ELF5 and CDX2. Larger groups of CDX2-positive cells are detected only in early gestation up to 8.5–9 weeks. CDX2 is mostly co-expressed with ELF5 (arrowheads). (C) Dual labelling of 6 week placental section for CDX2 and the proliferation marker Ki67. CDX2-expressing cytotrophoblasts preferentially stain positive for Ki67, indicating their high proliferation rate. CDX2 and Ki67 are restricted to the proximal end of cytotrophoblast cell columns (highlighted by the boxed area). The white arrows indicate the direction of progressive extravillous trophoblast (EVT) differentiation and migration.

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Expressing, Double Staining, Marker, Staining, Migration

Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 5. Inter-regulatory network of trophoblast transcription factors CDX2, EOMES and ELF5. (A) Chromatin immunoprecipitation assays show that CDX2 binds to the ELF5 promoter region in JEG-3 and TCL-1 cells where ELF5 is hypomethylated and expressed, but not in TCL-2 cells where ELF5 is hypermethylated and repressed. (B) In turn, ELF5 binds to the CDX2 and EOMES promoter regions in JEG-3 and TCL-1 cells where it is expressed, but not in TCL-2 cells from which it is absent, thereby establishing a transcrip- tional feedback loop between all three transcription factors. Binding to the EOMES promoter region was more consistent and is indicative of a more effi- cient, stronger interaction than with the CDX2 upstream region, consistent with results observed in mouse trophoblast (9).

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Chromatin Immunoprecipitation, Binding Assay

Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 6. Trophoblast transcription factor expression and epigenetic regulation of ELF5 in human ES cells and derived trophoblast cell lines. (A) Initial bisul- phite sequencing analysis of two pooled hES cell lines and derived trophoblast cells indicates a high degree of DNA methylation at the ELF5 promoter despite the limited trophoblast differentiation potential. (B) RT–PCR and (C) qPCR analysis for trophoblast transcription factors ELF5, CDX2 and EOMES on six differ- ent hES cells lines (Shef1, Shef4–7, H7), including one subclone with an abnormal karyotype (Shef5a), two derived cytotrophoblast cell lines (TrophH7 and TrophShef4), the JEG-3, TCL-1 and TCL-2 cell lines, an 8+4 week placenta for relative comparison of expression levels and a colorectal cancer cell line (DKO4) as positive control for CDX2 expression (27). Colour-inverted photographs of ethidium bromide stained gels are shown. ELF5 is detectable in some hES cell lines, albeit at very low levels. Higher expression levels of CDX2 and EOMES may relate to their function within the embryonic lineage and is not directly indicative of trophoblast differentiation potential. Strikingly, in contrast to their expression in placenta, all three genes are absent from the hES-derived tropho- blast cell lines. (D) Normalization of qPCR data to Shef6, one of the most highly ELF5 expressing hES cell lines, in comparison with JEG-3, TCL-1 and TCL-2 cell lines as well as a first trimester placenta sample demonstrates the comparatively negligible amount of ELF5 expression in hES cells that is approximately 300-fold less than in normal trophoblast in vivo. (E) Bisulphite sequencing analysis of the ELF5 promoter in three different hES cell lines and two derived trophoblast cell lines shows relatively little epigenetic variability between different hES cell lines. Hypermethylation correlates with extremely low ELF5 expression levels. (F) Elf5 is also highly methylated in three independent mouse epiblast stem cell lines and (G) in two human-induced pluripotent stem cell lines derived from kereatinocytes and fibroblasts.

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Expressing, Derivative Assay, Sequencing, DNA Methylation Assay, Reverse Transcription Polymerase Chain Reaction, Comparison, Positive Control, Staining, In Vivo, Bisulfite Sequencing, Methylation

Journal: Developmental Cell

Article Title: Maternal DNA Methylation Regulates Early Trophoblast Development

doi: 10.1016/j.devcel.2015.12.027

Figure Lengend Snippet: Scml2 Is Controlled by DNA Methylation and Affects SynT Formation and Cell Adhesion (A) RT-qPCR of Dnmt3a mKO EPCs confirms that Scml2 is controlled by oocyte methylation. (B) Methylation analysis by Sequenom MassARRAY in E7.5 male EPCs, confirming the DMR at an intragenic TSS of Scml2 . Each data point may include more than one CpG from the amplicon, as indicated on the x axis. (C) RT-qPCR analysis of TSCs grown in FGF+ (TSC conditions) or FGF− (differentiation conditions) medium for 6 days, with or without Scml2 overexpression. (D) Expression of Syna is reduced in Dnmt3a mKO EPCs, whereas markers of SynT-II Synb and Cebpa are unaffected. (E) E-cadherin staining of two independent Scml2 knockout clones from TKO TSCs shows a rescue of the morphological alterations seen in TKO TSCs. (F) Scml2 KO on TKO TSCs also rescues the defect in cell adhesion to cell culture wells in the absence of laminin. (G) RT-qPCR analysis of TKO Scml2 KO clones shows maintained expression of the TSC marker Cdx2 ; the expression of genes involved in cell adhesion is not rescued upon Scml2 deletion. Error bars represent SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; t test comparing WT and Dnmt3a mKO EPCs (A and D) or Scml2 -expressing TSCs versus vector control (C), or ANOVA with post hoc tests comparing Ctrl with DHet/DKO (B) or TKO TSC lines with WT TSCs. See also Figure S6 .

Article Snippet: Paraffin sections were deparaffinized with Histo-Clear and dehydrated through an ethanol series, followed either by standard H&E staining or antigen retrieval by boiling slides for 30 min (in 1 mM EDTA, 0.05% Tween 20, pH 8) and cooling at room temperature for 20 min. After blocking with 1% BSA overnight, sections were incubated with a rabbit monoclonal anti-CDX2 antibody (EPR2764Y, Novus Biologicals, 1:250 dilution) for 2 hr.

Techniques: DNA Methylation Assay, Quantitative RT-PCR, Methylation, Amplification, Over Expression, Expressing, Staining, Knock-Out, Clone Assay, Cell Culture, Marker, Plasmid Preparation, Control