Total proteins were extracted from tumor tissues and cells, and then separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). After transferring to polyvinylidene fluoride (PVDF) membrane and blocking with 5% bovine serum albumin (BSA), the PVDF membranes were probed at 4°C with rabbit anti-ALDH1 antibody (EP1933Y; 1:1,500, Abcam), cluster of differentiation (CD)133 (ab19898), nanog homeobox (Nanog) (ab109250), sex-determining region Y-Box transcription factor 2 (SOX2) (ab97959) oroctamer-binding transcription factor (Oct)4 (ab18976; 1:1,000, Abcam), E-cadherin (ab15148) or N-cadherin (ab76057; 1:2,000, Abcam), HMGA2 (1:2,500, Abcam), and GAPDH (ab9485; 1:3,000, Abcam) overnight. Following incubation with HRP-conjugated secondary antibodies (1:5,000, Abcam), the immunoreactivities were evaluated with enhanced chemiluminescence (KeyGen, Nanjin, China).
Ep1933y
Manufactured by Abcam
Sourced in United Kingdom
EP1933Y is a rabbit monoclonal antibody that recognizes the human FOXP3 protein. FOXP3 is a transcription factor that plays a key role in the development and function of regulatory T cells. The antibody is suitable for use in various applications, including flow cytometry, immunohistochemistry, and western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Ab15148, by Abcam (1 mentions)
Ab109250, by Abcam (1 mentions)
Ab18976, by Abcam (1 mentions)
Ab19898, by Abcam (1 mentions)
Enhanced chemiluminescence, by Abcam (1 mentions)
Ab97959, by Abcam (1 mentions)
Ab76057, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Abcam (1 mentions)
Ab9485, by Abcam (1 mentions)
Ab15289, by Abcam (1 mentions)
Startingblock t20 tbs blocking buffer, by Thermo Fisher Scientific (1 mentions)
Amersham hyperfilm ecl, by GE Healthcare (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Nupage 7 tris acetate gel, by Thermo Fisher Scientific (1 mentions)
Supersignal west dura chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Western lightning ultra, by PerkinElmer (1 mentions)
Protease phosphatase inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Hyblot cl autoradiography film, by Thomas Scientific (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
5 protocols using ep1933y
1
Profiling Stemness and EMT Markers
2
Western Blotting of Cell Lysates
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Cells were lysed in Cell Lysis Buffer (Cell Signaling Technology) supplemented with Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology). 30–50 μg of total protein lysate was run on Bolt 10% Bis-Tris Plus or NuPAGE 7% Tris-Acetate gels (Life Technologies) and transferred to PVDF membranes (Life Technologies). Membranes were blocked in StartingBlock T20 (TBS) blocking buffer (Thermo Scientific), and proteins were detected with the following antibodies: rabbit monoclonal Anti-C/EBP-β (Clone E299, Millipore), rabbit polyclonal Anti-FLI1 (ab15289, Abcam), rabbit polyclonal Anti-DAX1 (also known as NR0B1, sc-841, Santa Cruz Biotechnology), rabbit monoclonal Anti-ALDH1A1 (EP1933Y, Abcam), or mouse monoclonal Anti-GAPDH (Clone GAPDH-71.1, Sigma Aldrich) followed by HRP-anti-rabbit or HRP-anti-mouse secondary (GE Healthcare Life Sciences). Substrates used for detection were SuperSignal West Dura Chemiluminescent Substrate (Thermo Scientific) and Western Lightning Ultra (PerkinElmer). Signal was detected with a Bio-Rad ChemiDoc Gel Imager or developed on Amersham Hyperfilm ECL (GE Healthcare Life Sciences) or HyBlot CL Autoradiography Film (Denville Scientific). Bands were quantified using ImageJ.
3
Immunohistochemical Staining of ALDH1
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IHC was performed by using 4-µm thick, formalin-fixed, paraffin-embedded tissues. The tissues were dried overnight in an oven at 60℃, and placed in a Bond™ polymer refine detection system (Leica Biosystems Newcastle Ltd., Newcastle upon Tyne, UK). After deparaffinizing with Bond™ dewax solution (Leica Biosystems Newcastle Ltd.), pretreatment was performed by using Bond™ epitope retrieval solution 1 (Leica Biosystems Newcastle Ltd.) for 20 minutes at 98℃. Following this, the endogenous peroxidase was quenched by incubation with hydrogen peroxide for 15 minutes. Sections were incubated for 15 minutes at room temperature with the monoclonal antibody for ALDH1 (EP1933Y, 1:100; Abcam, Cambridge, UK) using biotin-free polymeric horseradish peroxidase-linker antibody conjugate system, and developed with 3,3 diaminobenzidine chromogen, in a Bond-maX™ automated slide stainer (Leica Biosystems Melbourne Pty. Ltd., Melbourne, Australia).
4
Immunohistochemical Profiling of FFPE Tumor Tissue
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Tumour tissue and the agarose-embedded close-to-patient cancer cells were formalin fixed and paraffin embedded (FFPE) before 4 μm sections were cut for both H&E and immunohistochemistry (IHC) analysis. IHC was performed using standard techniques and in line with the manufacturer's instructions for the following primary antibodies: Cytokeratin (MNF116, DAKO), EpCam (Ber-EP4, DAKO), CD44 (DF1485, DAKO), p53 (DO-7, DAKO), Vimentin (V9, DAKO), TFF3 (Abcam), and ALDH1A1 (EP1933Y, Abcam) (see online supplementary method S6). Sections were viewed with a Leica DMLB Bright-field Microscope (Leica-microsystems, Milton Keynes, UK) and images acquired with Leica QWin Standard v3 software. The presence of any characteristically stained cells was considered positive with respect to negative controls, and confirmed by a second blinded individual.
5
ALDH1A1 Expression in TNBC Patients
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Patients and samples. Tissue samples were obtained from patients who underwent surgery at the Yokohama City University Medical Center. Five patients with triple-negative breast cancer (TNBC) and ALDH1A1 expression were enrolled in this study. The patients did not receive any preoperative treatments to avoid potential gene modification. This study was approved by the Institutional Review Board of Yokohama City University (D1207027). All procedures performed on human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The patients provided informed consent prior to inclusion in the study.
Histopathological and immunohistological staining. Hematoxylin and eosin (H&E)-stained sections from each block were prepared to determine the histological examination and diagnosis. To determine the breast cancer subtype, immunohistochemistry (IHC) of paraffinembedded breast cancer tissues was performed to detect ER, PgR, and HER2. ER-negative, PgR-negative, and HER2-negative tumors were considered as TNBC. IHC was performed with an anti-ALDH1A1 (EP1933Y, ab52492, Abcam, Cambridge, UK) antibody. The IHC protocol with anti-ALDH1A1 was as previously described (4) (link). Representative images of the H&E and ALDH1A1 staining are shown in Figure 1.
Histopathological and immunohistological staining. Hematoxylin and eosin (H&E)-stained sections from each block were prepared to determine the histological examination and diagnosis. To determine the breast cancer subtype, immunohistochemistry (IHC) of paraffinembedded breast cancer tissues was performed to detect ER, PgR, and HER2. ER-negative, PgR-negative, and HER2-negative tumors were considered as TNBC. IHC was performed with an anti-ALDH1A1 (EP1933Y, ab52492, Abcam, Cambridge, UK) antibody. The IHC protocol with anti-ALDH1A1 was as previously described (4) (link). Representative images of the H&E and ALDH1A1 staining are shown in Figure 1.
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