Anti-vimentin, anti-Snail, and anti-Slug antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-NUCB2, anti-Twist1, and anti-Ki-67 antibodies were obtained from Abcam (Cambridge, MA, USA). Anti-E-cadherin, anti-p21waf1, anti-BCL2, and anti-cyclin D1 antibodies were from Dako (Copenhagen, Denmark). Anti-p27kip1, anti-Rb, anti-X-linked inhibitor of apoptosis (XIAP), anti-BAX, and anti-N-cadherin antibodies were from BD Biosciences (San Jose, CA, USA). Anti-ZEB1 and anti-β-actin antibodies and adriamycin (ADR: D1515) were from Sigma-Aldrich Chemicals (St. Louis, MO, USA). Anti-phospho (p) Rb at Ser807/811, anti-cleaved caspase-3, and anti-poly (ADP-ribose) polymerase 1 (PARP1) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-cyclin A2 and anti-cyclin B1 antibodies were from Novocastra (Newcastle, UK) and Santa Cruz Biotech (Santa Cruz, CA, USA), respectively.
Anti e cadherin
Manufactured by Agilent Technologies
Sourced in Denmark, United States
Anti-E-cadherin is a primary antibody that binds to the extracellular domain of E-cadherin, a cell-cell adhesion protein. It is commonly used in research applications to detect and quantify E-cadherin expression levels in various cell types and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Anti p21waf1, by Agilent Technologies (3 mentions)
Anti p27kip1, by BD (3 mentions)
Anti vimentin, by Agilent Technologies (3 mentions)
Anti vimentin, by Cell Signaling Technology (2 mentions)
Anti slug, by Cell Signaling Technology (2 mentions)
Anti cyclin d1, by Agilent Technologies (2 mentions)
Anti phospho rb at ser807 811, by Cell Signaling Technology (2 mentions)
Anti poly adp ribose polymerase 1 parp 1, by Cell Signaling Technology (2 mentions)
Anti cyclin a2, by Leica (2 mentions)
Anti n cadherin, by BD (2 mentions)
Anti β catenin, by Cell Signaling Technology (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti ki67, by Abcam (1 mentions)
Anti snail, by Cell Signaling Technology (1 mentions)
Anti twist1, by Abcam (1 mentions)
Anti bcl2, by Agilent Technologies (1 mentions)
Anti rb, by BD (1 mentions)
Anti zeb1 and anti β actin antibodies, by Merck Group (1 mentions)
Anti cyclin b1, by Leica (1 mentions)
Vimentin, by Merck Group (1 mentions)
13 protocols using anti e cadherin
1
Antibody Acquisition for EMT Analysis
2
Profiling Heat-Induced Molecular Changes
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Changes in the molecular properties of the heat-exposed cells were investigated using ICC, western blotting, and qRT-PCR. Five different primary antibodies (DAKO, Denmark), anti-E-cadherin (1 : 300), anti-CK8/18 (1 : 200), anti-vimentin (1 : 1000), anti-desmin (1 : 150), and anti-p53 (1 : 300), were employed for immunocytochemistry (ICC). The antibodies were applied to quantify the changes in EMT-related properties or demonstrate the cell death mechanism.
The changes in protein expression were further determined by western blotting. The expression of heat shock protein 70 (Hsp70, 1 : 1000, Merck Millipore, Germany), Hsp90 (1 : 500, Santa Cruz, CA, USA), E-cadherin (41 : 1000, Merck Millipore), and vimentin (1 : 1000, Merck Millipore) were quantified by comparing to that of β-actin (1 : 1000, Merck Millipore).
EMT-related genes were quantified using qRT-PCR. Primer sequences, including HSPA1A (Hs00359163_s1), CDH1 (Hs01023894_m1), VIM (Hs00958111_m1), TWIST1 (Hs01675818_s1), and GADPH (Hs02758991_g1), were purchased from Thermo Fisher Scientific. See ESI† for additional details.
The changes in protein expression were further determined by western blotting. The expression of heat shock protein 70 (Hsp70, 1 : 1000, Merck Millipore, Germany), Hsp90 (1 : 500, Santa Cruz, CA, USA), E-cadherin (41 : 1000, Merck Millipore), and vimentin (1 : 1000, Merck Millipore) were quantified by comparing to that of β-actin (1 : 1000, Merck Millipore).
EMT-related genes were quantified using qRT-PCR. Primer sequences, including HSPA1A (Hs00359163_s1), CDH1 (Hs01023894_m1), VIM (Hs00958111_m1), TWIST1 (Hs01675818_s1), and GADPH (Hs02758991_g1), were purchased from Thermo Fisher Scientific. See ESI
3
Protein Extraction and Immunoblotting Protocol
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Protein extraction and immunoblotting were performed as previously described [47 (link)]. Primary antibodies were as follows: anti-Notch1 (Clone A6 Novus Biologicals, Cambridge, UK), anti-Thbs1(sc-73158, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-E-Cadherin (Clone NCH-38, Dako, Denmark) anti-mTor (2993, Cell Signaling Technology, Beverly, MA), anti-Icam5 (Abcam, Cambridge, UK), anti Pai3 (sc-99153, Santa Cruz Biotechnology), anti-Vimentin (Clone V9, Dako), anti Ck19 (Clone RCK108, Dako), anti-Ck18 (Clone DC10, Dako), anti-Mmp-9 (Clone 6-6B, Calbiochem, San Diego, USA), anti-Snail (sc-28199, Santa Cruz Biotechnology), anti-Ck8 (sc-52324, Santa Cruz Biotechnology), anti-Alpha-Sma (Clone 1A4, Sigma), anti-E-Cadherin (Clone 4A2, Cell Signaling) and anti-β-Actin monoclonal antibody (Clone AC-40). Immunoreactivities were revealed with the EnVision dextran polymer visualization system (Dako). Membranes were washed and autoradiographies were obtained using a chemiluminescence reaction (ECL reagents, Amersham). Digital images of autoradiographies were acquired with a scanner (Fluor-S MultiImager, Bio-Rad) and signals were acquired in the linear range of the scanner and quantified using specific densitometric software (QUANTITY-ONE, Bio-Rad) in absorbance units.
4
Characterization of Breast Cancer Cell Lines
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BT474, T47D, MDA-MB-231, BT549 and SKBR3 breast cancer cell lines were purchased from the Committee on Type Culture Collection of the Chinese Academy of Science (Shanghai, China). The pEGFP-C1-ERα plasmid was purchased from Addgene (Cambridge, MA, USA), and pcDNA3-BMI1 plasmid was a gift from Dr. MH Yang (Institute of Clinical Medicine, National Yang-Ming University, Taipei city, Taiwan) [20 (link)]. A wild type BMI1 promoter (pXP2-BMI1-Luc1000) from −1023 to −1 was constructed and a mutant BMI1 promoter was generated by replacing a TGACC (−178–174) sequence in the wild type BMI1 promoter with a GACCC sequence. The following antibodies were used in this study: anti-E-cadherin (DAKO, Glostrup, Denmark; NCH-38), anti-Bmi1 (Abcam, Cambridge, UK; ab126783), anti-ERα (Santa Cruz Biotechnology, CA, USA; sc-543), anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA; sc-4777), PE-conjugated anti-CD24 (BD Biosciences, Bedford, MA, USA; ML5), APC-conjugated anti-CD44 (BD Pharmigen, San Jose, CA, USA; G44-26), PE-conjugated mouse IgG (BD; G155-178), and APC-conjugated mouse IgG (BD; 27–35).
5
Immunohistochemistry Staining Protocol
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Immunohistochemical studies were performed on formalin-fixed, paraffin-embedded sections as previously described [19 (link), 40 (link)], or in a Dako AutoStainer (Dako, Carpinteria, CA). Antibodies used include: anti-CD68 (clone PG-M1, Dako, dilution 1:100); anti-CD163 (clone10D6, Biocare Medical, Concord, CA, USA, dilution 1:50); anti-CD31 (clone JC70A, Dako, ready-to use); anti-MMP-9 (clone Ab-2, Oncogene Research Products Cambridge, MA, dilution 1:50); and anti E-cadherin (Dako, clone NCH-38, ready to use).
6
Immunohistochemical analysis of TLR7, E-cadherin, and Vimentin
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Human tumor samples were fixed in neutral-buffered 10% formalin solution and paraffin embedded. TLR7 staining and quantification was performed as previously described 11 using TLR7-specific polyclonal antibody (ENZO Lifesciences) at 10 mg/mL.
The expression of E-cadherin (Dako M3613, 1/100 dilution) and vimentin (Cells Signaling 5741, 1/100) by tumor cells was performed as follows. Serial 5-μm tissue sections were deparaffinized, rehydrated, and pretreated in 10 mM citrate buffer, pH 6, for antigen retrieval. Sections were incubated with hydrogen peroxide for 15 minutes, then blocked in 5% human serum for 30 minutes at room temperature. The slides were then incubated with a primary antibody (diluted in Dako real solution, antibody diluent) anti-E-cadherin (Dako M3613, 1/100 dilution) for one hour at room temperature or anti-vimentin (Cells Signaling 5741, 1/100) overnight. Slides were then incubated for 30 minutes at room temperature with the secondary antibody (anti-rabbit coupled to biotin, 1/200, JIR 715–066-150), followed by 30 minutes incubation with streptavidin-HRP (Dako, 1/300). After each incubation, the slides were washed 5 minutes with 1x TBS+ 0.04% Tween. Revelation was performed with the DAB kit (Dako, K3468) and stopped by placing slides in 1X TBS and distillated water. Counter coloration was performed with hematoxylin.
The expression of E-cadherin (Dako M3613, 1/100 dilution) and vimentin (Cells Signaling 5741, 1/100) by tumor cells was performed as follows. Serial 5-μm tissue sections were deparaffinized, rehydrated, and pretreated in 10 mM citrate buffer, pH 6, for antigen retrieval. Sections were incubated with hydrogen peroxide for 15 minutes, then blocked in 5% human serum for 30 minutes at room temperature. The slides were then incubated with a primary antibody (diluted in Dako real solution, antibody diluent) anti-E-cadherin (Dako M3613, 1/100 dilution) for one hour at room temperature or anti-vimentin (Cells Signaling 5741, 1/100) overnight. Slides were then incubated for 30 minutes at room temperature with the secondary antibody (anti-rabbit coupled to biotin, 1/200, JIR 715–066-150), followed by 30 minutes incubation with streptavidin-HRP (Dako, 1/300). After each incubation, the slides were washed 5 minutes with 1x TBS+ 0.04% Tween. Revelation was performed with the DAB kit (Dako, K3468) and stopped by placing slides in 1X TBS and distillated water. Counter coloration was performed with hematoxylin.
7
Comprehensive Antibody Inventory for Cell Analysis
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Anti‐Ezrin/radixin/moesin (ERM), anti‐phospho (p) Ezrin (at Thr567)/radixin (at Thr564)/moesin at Thr558) (pERM), anti‐poly (ADP‐ribose) polymerase 1 (PARP1), and anti‐vimentin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti‐EBP50, anti‐β‐actin, anti‐MYH9 (mouse), anti‐vinculin, and anti‐Sox2 antibodies were obtained from Abcam (Cambridge, MA, USA). Anti‐E‐cadherin, anti‐p21waf1, and anti‐cyclin D1 antibodies were from Dako (Copenhagen, Denmark). Anti‐cyclin A2 and anti‐cyclin E antibodies were from Novocastra (Newcastle, UK). Anti‐p27kip1, anti‐aldehyde dehydrogenase 1 (ALDH1), and anti‐N‐cadherin antibodies were from BD Biosciences (San Jose, CA, USA). Anti‐MYH9 (Rabbit) and anti‐cyclin B1 antibodies were purchased from Proteintech (Rosemont, IL, USA) and Santa Cruz Biotech (Santa Cruz, CA, USA), respectively. Blebbistatin and MG132 were obtained from Toronto Research Chemicals (North York, ON, Canada) and Sigma‐Aldrich Chemicals (St. Louis, MO, USA), respectively.
8
Immunohistochemical Analysis of Immune Markers
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Immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded sections using following antibodies and dilution ratios: rat monoclonal anti-mouse F4/80 antibody (1:250, Abcam, Cambridge, UK), rabbit polyclonal anti-mouse CD3 antibody (1:500, Abcam), and rat monoclonal anti-mouse E-cadherin (1:200, eBioscience). Briefly, after dewaxing and rehydration, a heat-inducing antigen retrieval procedure using citrate buffer at pH 6.0 for 21 min was performed on all tissue sections, with subsequent washing in PBS and endogenous peroxidase blocking with EnVisionTM FLEX Peroxidase-Blocking Reagent (Dako, Agilent, Santa Clara, CA, USA) for 10 min. Sections were incubated with primary antibodies overnight. Sections stained with anti-F4/80 and anti-E-cadherin antibody were incubated with secondary polyclonal rabbit anti-rat immunoglobulins/HRP (1:500, Dako) for 60 min. The CD3 sections were treated by applying the commercial EnVisionTM FLEX/HRP detection reagent (Dako). Immunoreactions were developed with diaminobenzidine (DAB, Dako) diluted in EnVisionTM FLEX Substarte Buffer (Dako). The sections were counterstained with haematoxylin.
9
Immunohistochemical Assessment of Epithelial-Mesenchymal Transition Markers
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Paraffin-embedded slides were incubated with primary antibodies: anti-Ube2v1 (Abcam, polyclonal ab88679), anti-E-cadherin (Dako, monoclonal M3612), anti-Fibronectin (Boster, polyclonal BA1771), anti-Vimentin (Abcam, monoclonal, ab8978), anti-β-catenin (Cell Signaling Technology, monoclonal #8480), anti-SQSTM1/p62 (CST, polyclonal 5114), and anti-Beclin1 (Boster, polyclonal PB0014). Staining was done on a SPlink Detection Kit (SP-9000). We quantified staining intensity and percentage of stained cells. Positive tumor cells were quantified by two independent observers. The staining intensity was scored on a scale of 0–3 as negative (0), weak (1), medium (2), or strong (3). The extent of the staining, defined as the percentage of positive staining areas of tumor cells in relation to the whole tumor area, was scored on a scale of 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). An overall protein expression score (overall score range, 0–12) was calculated by multiplying the intensity and extent positively scores.
10
Immunofluorescence Staining of Cell-Cell Junctions
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Samples were deparaffinized and rehydrated. To unmask antigens, citrate buffer (pH 6.0) was used in a Biocare Company Decloaking Unit at 110°C for 15 min followed by TBST washing (5 min×2) and blocking in 5% normal goat serum (Cell Signaling). Anti-ZO1 (Invitrogen, rabbit) 1:100 in Antibody Diluent (Cell Signaling, #8112) and anti-E-cadherin 1:100 (Dako, M3612, mouse) were then used. Sections were incubated with goat anti-rabbit Alexa Fluor 488 at 1:500 and goat anti-mouse Alexa Fluor 568 at 1:500 (Invitrogen) for 45 min at room temperature, washed 5 min×3 with PBS, and slides were mounted with hard-set fluorescence mounting medium (Vector Laboratories). Images were captured using confocal microscopy.
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