Cells were cultured on glass coverslips and treated with 250 µM UDCA, 10 nM gefitinib, or 50 nM EGF for 24 h. Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich; Merck KGaA) for 10 min, and then, cells were permeabilized with 0.3% triton x-100 in PBS for 10 min. Cells were incubated further with 10% goat serum (Cat. #sc-2043) (Santa Cruz Biotechnology) solution at room temperature for 1 h. Cells were incubated with an E-cadherin antibody (1:200 dilution, Cat. #3195; Cell Signaling Technology) in PBS containing 1% BSA (PBS-A) at room temperature. After reaction, this was incubated further with 1% albumin for 1 h and then goat serum solution at room temperature for 1 h. The cells were treated with a FITC-conjugated secondary antibody (1:500 dilution, Cat. #sc-36869, Santa Cruz Biotechnology) for 1 h in PBS-A and rinsed with PBS three times. The cells on coverslips were then treated with DAPI (0.5 µg/ml) (Sigma-Aldrich; Merck KGaA) for 1 min, and images were captured (×400 optical and ×3 digital magnification) using a super-resolution confocal laser microscope (Carl Zeiss).
E cadherin antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The E-cadherin antibody is a laboratory reagent used for the detection and analysis of the E-cadherin protein. E-cadherin is a cell adhesion molecule that plays a crucial role in the formation and maintenance of cell-cell junctions. This antibody can be used in various applications, such as immunohistochemistry, Western blotting, and flow cytometry, to investigate the expression and localization of E-cadherin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
N cadherin antibody, by Santa Cruz Biotechnology (3 mentions)
Vimentin antibody, by Santa Cruz Biotechnology (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Fitc conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Goat serum, by Santa Cruz Biotechnology (1 mentions)
Alexa 647, by Thermo Fisher Scientific (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Gapdh antibody, by Santa Cruz Biotechnology (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Merck Group (1 mentions)
Fibroblast growth factor (fgf), by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Fujifilm (1 mentions)
17 protocols using e cadherin antibody
1
E-cadherin Localization in UDCA, Gefitinib, and EGF Treated Cells
2
Quantifying Induced E-Cadherin Membrane Expression
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HEK293T cells were plated on 6 well plates for 24 h prior to use. Human E-cadherin-GFP and inducible zebrafish WT or mutant march8 plasmids were transfected into the cells (see above). Cells were incubated for 12 h and then transferred to fresh media containing 50 µM tebufenozide for 8 h. After incubation, the cells were harvested and non-permeablized cells were stained at 4°C with extracellular domain-specific E-cadherin antibody (67A4, Santa Cruz, 1∶100) for 30 min in FACS buffer (0.5% BSA, 1% Sodium Azide in PBS). Cells were washed 3 times with FACS buffer and were stained with Alexa-647 (1∶1000, Molecular Probe) secondary antibody at 4°C for 30 min. Data acquisition was performed on a FACScan flow cytometer (BD Bioscience), and data were analyzed using FloJo software.
3
Comprehensive Reagent Procurement for Cell Culture
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Crystal violet, sodium dodecyl sulfate (SDS), bovine serum albumin (BSA), insulin, Triton X-100, trypsin, cordycepin, and trypan blue were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Moreover, fetal bovine serum (FBS) was purchased from Life Technologies (Auckland, New Zealand), and dimethyl sulfoxide was purchased from Wako Pure Chemical Industries (Saitama, Japan). Vimentin antibody, E-cadherin antibody, and GAPDH antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). The PGC-1alpha antibody was obtained from Novus (Littleton, CO, USA). TGF-beta was purchased from Preprotech (London, UK). CD44-FITC antibody and CD117-PE antibody were purchased from (eBioscience, CA, USA). Epidermal growth factor and fibroblast growth factor were purchased from Invitrogen (CA, USA).
4
Isolation of E-cadherin-positive and -negative cells
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TGFβ3 treated cells were prepared as described before. Cells (10 × 106) were harvested, washed in FACS buffer (1 mM EDTA, 25mM HEPES and 1% FBS in PBS), and then stained with E-cadherin antibody (sc-8426, Santa Cruz Biotechnology, Dallas, TX, USA) and goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 488 (A-11001, Thermo Fisher Scientific, Waltham, MA USA) for 30 min at 4 °C. Cells were washed with FACS buffer and stained with DAPI (final concentration: 0.1 µg/µL). Stained cells subsequently collected by BD FACSAria™ III and single E-cadherin-positive and E-cadherin-negative cells were collected in individual wells of a 96-well plate and allowed to grow into colonies.
5
Regulation of EMT by GROα in Tumor Cells
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Materials were purchased from various manufacturers: recombinant GROα (R&D Systems), GROα siRNA (Santa Cruz Biotechnology), control siRNA (Santa Cruz Biotechnology), Lipofectamine ltx (Invitrogen), Opti-MEM Reduced Serum medium (Invitrogen), GROα ELISA kit (R&D Systems), GROα primers (IDT), ThermoScript™ RT-PCR systems (Invitrogen), SYBR Green (Qiagen), EMT marker primers (IDT), PD98059 (Cell Signaling), E-cadherin antibody (Santa Cruz Biotechnology), N-cadherin antibody (Santa Cruz Biotechnology), Snail antibody (Abcam), Slug antibody (Abcam), Twist antibody (Santa Cruz Biotechnology), Vimentin antibody (Santa Cruz Biotechnology), VEGF antibody (Santa Cruz Biotechnology), phospho-MAPK antibody (Cell Signaling), MAPK antibody (Cell Signaling), β-actin mouse antibody (Santa Cruz Biotechnology).
6
Immunohistochemical Analysis of Intestinal E-Cadherin and Occludin
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After 7 days of feeding, the small intestine from experimental mice was removed for histological studies. Deparaffinized and rehydrated 5-μm tissue sections were rinsed with PBS, permeabilized with 0.1% Triton for 5 min and blocked for 10 min with 0.1% H2O2 to inhibit endogenous peroxidase activity. After rinsing in PBS, the sections were blocked with bovine serum albumin (1% BSA) for 30 min and then with normal goat serum (1/100).
The sections were incubated with the corresponding primary antibodies: E-cadherin antibody (sc-8426 Santa Cruz, Biotechnology) and occludin antibody (AA 480–520 antibodies-online Inc.) at 4 °C, overnight. Afterward, they were rinsed with PBS, and incubated at room temperature (RT) for 1 h with the secondary antibody peroxidase goat anti-rabbit IgG (Horseradish Peroxidase—HRP) (ab6721). Then the slices were incubated with a peroxidase (HRP) detection system (DAB Peroxidase Substrate. SK-4100 VECTOR Laboratories) at RT for 2–12 min and washed for 5 min in water. A counterstain was conducted with hematoxylin, before optical microscopy observation.
The sections were incubated with the corresponding primary antibodies: E-cadherin antibody (sc-8426 Santa Cruz, Biotechnology) and occludin antibody (AA 480–520 antibodies-online Inc.) at 4 °C, overnight. Afterward, they were rinsed with PBS, and incubated at room temperature (RT) for 1 h with the secondary antibody peroxidase goat anti-rabbit IgG (Horseradish Peroxidase—HRP) (ab6721). Then the slices were incubated with a peroxidase (HRP) detection system (DAB Peroxidase Substrate. SK-4100 VECTOR Laboratories) at RT for 2–12 min and washed for 5 min in water. A counterstain was conducted with hematoxylin, before optical microscopy observation.
7
Immunoblotting and Protein Detection Protocol
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Immunoblotting and protein detection were essentially performed as previously described [61 (link)]. Protein detection was performed using the following primary antibodies; mouse monoclonal E-cadherin antibody (Santa Cruz, sc-8426), anti-GAPDH (Abcam, ab9484), anti-Myc (Santa Cruz, sc-40), anti-α-tubulin (Sigma, T5168), anti-β-actin (Sigma-Aldrich, A5441), anti-FOXM1 (Santa Cruz, sc-271746); and rabbit polyclonal anti-KIF4A (Invitrogen, PA5-30492), anti-NTCP (Sigma, HPA042727), anti-HA (Sigma, H6908). For immunoblotting of free or tagged NTCP, the sample was treated with 250-U Peptide-N-Glycosidase F (PNGase F) to digest N-linked oligosaccharides from glycoproteins before loading to SDS-PAGE [27 (link)].
8
Immunohistochemical Evaluation of MET, HGF/SF, and Proliferation Markers
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To determine MET expression status by immunohistochemistry (IHC), monoclonal antibody (mAb) for MET, MET4 mAb,26 was used. Epitope sequence of MET4 revealed that DVLPEFR on amino acid residue 236–242 was the specific recognition site of MET. To retrieve MET antigen, tissue slide sections were treated at 98°C for 20 min in Target Retrieval Solution, pH9 (DAKO, Santa Clara, CA). ENVISION+ System (DAKO) was used for the secondary antibody reaction and DAB+ was used for color development. In the final step, the nuclei were lightly counterstained with Meyer hematoxylin.
Regarding the tissue staining of HGF/SF, anti‐HGF/SF mAb (clone 7‐2) was purchased from Enzo Life Sciences (Ann Arbor, MI, USA) and the samples were stained according to the company's instruction. Antigen retrieval was performed by boiling the sample slides in the 10 mM citrate buffer (pH 6) in a microwave for 7–10 min.
For the evaluation of Her2/neu IHC status HercepTest II (DAKO) was used. For the evaluation of tumor cell proliferation, we performed Ki‐67 staining using anti‐human Ki‐67 antibody (M7240, DAKO). E‐cadherin expression in the proliferative margin of tumor cells was also investigated using E‐cadherin antibody (H‐108, Santa Cruz Biotechnology, CA, USA).
Regarding the tissue staining of HGF/SF, anti‐HGF/SF mAb (clone 7‐2) was purchased from Enzo Life Sciences (Ann Arbor, MI, USA) and the samples were stained according to the company's instruction. Antigen retrieval was performed by boiling the sample slides in the 10 mM citrate buffer (pH 6) in a microwave for 7–10 min.
For the evaluation of Her2/neu IHC status HercepTest II (DAKO) was used. For the evaluation of tumor cell proliferation, we performed Ki‐67 staining using anti‐human Ki‐67 antibody (M7240, DAKO). E‐cadherin expression in the proliferative margin of tumor cells was also investigated using E‐cadherin antibody (H‐108, Santa Cruz Biotechnology, CA, USA).
9
Transfection and Expression of GLI1 and Cav-1
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DMEM medium, RPMI 1640 medium, FBS and trypsin/EDTA were purchased from Invitrogen Co. (Carlsbad, CA, USA). The GLI1 expressing plasmid was constructed by cloning GLI1 cDNA into the pCMV-Tag2B vector from Stratagene (Santa Clara, CA). Cav-1 cDNA was cloned into the pCMV-Tag2B vector to construct Cav-1 expressing plasmid. Both GLI1 siRNAs (Catalog No.: sc-37911) and Cav-1 siRNAs (Catalog No.: sc-29241) were from Santa Cruz Biotechnology, Santa Cruz, CA, USA). The 18s rRNA TaqMan probe (Hs99999901_s1), Cav-1 TaqMan probe (Hs00971716_m1) and GLI1 TaqMan probe (Hs01110766_m1) were purchased from Applied Biosystems (Carlsbad, CA, USA). The rabbit polyclonal GLI1 antibody, rabbit polyclonal Cav-1 antibody, rabbit polyclonal SNAI1 antibody, rabbit polyclonal Slug antibody and rabbit polyclonal PTCH1 antibody were from Cell signaling (Danvers, MA, USA). The mouse monoclonal E-cadherin antibody and rabbit polyclonal N-cadherin antibody were both purchased from Santa Cruz Biotechnology. The rabbit polyclonal Twist antibody was from Abcam (MA, USA). The rabbit polyclonal Fibronectin antibody, mouse monoclonal Vimentin antibody, mouse monoclonal β-actin antibody and 4,6-diamidino-2-phenylindole (DAPI) were from Boster Biotechnology (Wuhan, China).
10
Characterization of Human Hepatocellular Carcinoma Cell Lines
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Human HCC cell lines MHCC97-H, SMMC-7721, HepG2, and Huh7 were obtained from Shanghai cellular biology center, MHCC97-H cells labeled with luciferase (MHCC97-H-luc) were kindly provided by Prof. Yong Chen and Dr. Yang Cao from our laboratory. Cells were cultured in DMEM (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), penicillin (100 units/ml) and streptomycin (100 mg/ml) and incubated at 37°C with 5% CO2. Phosphate-buffered saline (PBS) with 0.25% trypsin and 0.01% EDTA was used for cell harvesting and passage. Antibodies for CD44, Vimentin, N-cadherin, ERK, and p-ERK (Thr202/Ty204) were purchased from Cell Signaling Technologies (Beverly, MA, USA). E-cadherin antibody was purchased from Santa Cruz (Santa Cruz, CA, USA). Antibodies for β-actin and Snail were purchased from Abcam (Cambridge, UK).
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