Participants who provided samples also provided written, informed consent to participate in this study. The Ethics Committee of the Shanghai Cancer Institute approved the study, the consent procedure, and the tissue array study. All of the research was performed in China. Paired colorectal tumor tissues and their corresponding adjacent non-tumor colorectal tissues (5 cm away from the lesions) were collected from patients who underwent curative surgery for CRC at Anhui Medical University, Anhui Province, China. Normal colon tissue was collected from patients with non-cancerous colon disease. A CRC diagnosis was confirmed by histological examination, and the relevant clinical and pathological information was retrieved from the hospital database (Additional file
1 : Table S1a). Glass-slide tissue arrays for CRC were purchased from the Shanghai Outdo Biotech Co., Ltd. (Shanghai, China) (Additional file
1 : Table S2a), and immunostaining (SOX2, ab75485, 1:100, Abcam, Cambridge, MA; vimentin, #5741, 1:50, Cell Signaling Technology, Beverly, MA) was performed on the tissue microarray slides. Staining was analyzed based on the percentage of positively stained cells and staining intensity by a pathologist or using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Bethesda, MD) (Additional file
2 ).
Vimentin 5741
Manufactured by Cell Signaling Technology
Sourced in United States, China, United Kingdom
Vimentin (5741) is a primary antibody that recognizes the vimentin protein. Vimentin is a type III intermediate filament protein that is expressed in various cell types, including mesenchymal cells and some epithelial cells. This antibody can be used to detect and study the expression and localization of vimentin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
E cadherin 3195, by Cell Signaling Technology (6 mentions)
S2367, by Agilent Technologies (2 mentions)
K3468, by Agilent Technologies (2 mentions)
Pk6106, by Vector Laboratories (2 mentions)
Inhibin βa hpa020031, by Merck Group (2 mentions)
Dp72 8 bit rgb camera, by Olympus (2 mentions)
X0909, by Agilent Technologies (2 mentions)
Zeb1 3396, by Cell Signaling Technology (2 mentions)
Gapdh fl 335, by Santa Cruz Biotechnology (2 mentions)
Bip 3183, by Cell Signaling Technology (2 mentions)
Eif2α 9722, by Cell Signaling Technology (2 mentions)
Tubulin b512, by Merck Group (2 mentions)
Alexa fluor 488 goat anti rabbit igg a11034, by Thermo Fisher Scientific (2 mentions)
Snail, by Cell Signaling Technology (2 mentions)
P eif2α, by Cell Signaling Technology (2 mentions)
Alexa fluor 568 phalloidin, by Thermo Fisher Scientific (2 mentions)
β catenin, by Cell Signaling Technology (2 mentions)
N cadherin 13116s, by Cell Signaling Technology (2 mentions)
E cadherin ab15148, by Abcam (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
24 protocols using vimentin 5741
1
Colorectal Cancer Tissue Analysis
2
Comprehensive Histopathological Analysis of Prostate Tumors
3
Immunohistochemical Analysis of Breast Tissue Microarrays
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Assembly and IHC staining of human breast tissue microarrays (TMAs) were performed by the Biorepository and Tissue Analysis core at MUSC. TMAs consisted of matched normal and tumor tissue as well as normal and metastatic lymph node biopsies from patients for which survival data was available (Supplementary Table S2 ). For IHC staining of formalin-fixed, paraffin-embedded TMAs and mouse lung tissue, sections were deparaffinized in xylene, rehydrated in alcohol, and processed as follows: The sections were incubated with target retrieval solution (S2367; Dako, Carpinteria, CA, USA) in a steamer for 45 min followed by 3% hydrogen peroxide solution for 10 min and protein block (X0909; Dako) for 20 min at room temperature. Sections were incubated overnight in a humid chamber at 4°C with antibodies against Inhibin βA [HPA020031] (1:3200; Sigma), E-cadherin [#3195], Vimentin [#5741] or Ki-67 [#12202] (1:400; Cell Signaling) followed by biotinylated secondary antibody (PK6106; Vector laboratories, Burlingame, CA, USA) for 30 min and ABC reagent for 30 min. Immunocomplexes of horseradish peroxidase were visualized by DAB reaction (K3468; Dako), and sections were counterstained with hematoxylin before mounting. Micrographs of stained sections were taken using an Olympus DP72 8-bit RGB camera with Cellsens acquisition software.
4
Protein Expression Analysis via Western Blotting
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Western blots was used to evaluate the expression of proteins. Cells (2 × 106) were seeded onto 100-mm Petri dishes and cultured until reached 90% of confluence. Then, cells were induced as described above. After 24 h, cells were scratched, and protein was isolated with RIPA buffer supplemented with phosphatase and protease inhibitors, as well as PMSF. The concentration of protein was determined with Direct Detect® (Merck Millipore, Burlington, MA, USA). The probes of three replicates were mixed together, and 30 µg of protein was used for electrophoresis and then transferred onto poly vinylidene fluoride (PVDF, Sigma Aldrich, St. Louis, MO, USA) membranes with wet transfer (400 mA, 110 min). Primary antibodies (Cell Signaling Technology, Leiden, Holland) were used: E-kadherin #3195, vimentin #5741, Akt #9272, phospho-Akt (Ser473) #4060, p44/42 #4695, phospho-p44/42 (Thr2020) #4370, cleaved-PARP1 (Asp214) #5625, SOD1 #4266, SOD2 #13141. GAPDH antibody (sc-32233, Santa Cruz Biotechnology, Heidelberg, Germany) was used as reference. Secondary antibodies conjugated with alkaline phosphatase (Sigma Aldrich) were used. Densitometry analysis was conducted in ImageJ program (ImageJ software, https://imagej.nih.gov/ij/ , NIH).
5
Epithelial-Mesenchymal Transition Markers
6
Immunohistochemical Analysis of Colorectal Tissues
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Colorectal tissues were fixed in 4% polyformaldehyde followed by paraffin embedding and then the sections were deparaffinized, rehydrated and immersed in 10 mM citrate buffer for heat-induced antigen retrieval. The tissues were stained with antibodies against CXCR7 (ab72100) (Abcam, UK), Vimentin (5741) (Cell Signaling Technology, USA) and YAP1 (13584-1-AP) (Proteintech, USA) (1:200 dilution) in accordance with manufacturer’s suggestions. The staining results were scanned using a digital slide scanning system (Pannoramic Scan, MedicalEXPO, France) and semi-quantified of mean density (IOD/area) by Image-Pro Plus 6.0 software (IPP).
7
Western Blot Analysis of TGF-β Signaling
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Fresh hepatocytes and AS-30D cells were lysed with RIPA buffer plus a cocktail of protease inhibitors (cOmplet protease Inhibitors, Roche, Basel, CHE) and phosphatase inhibitors (1 mM NaF, 1 mM NaVO3); 100 μg of total protein extracts were run in SDS-PAGE as described before (36 (link)). Antibodies used were the following ones: vimentin 5741, pSmad2 3108 (Cell Signaling Technology, Danvers, MA, USA), osteopontin AF808 (R&D Systems, Minneapolis, MN, USA), PMCA 1-4 sc-20028 (Santa Cruz Biotechnology, Dallas, TX, USA), and β-actin A2228 (Sigma-Aldrich, St. Louis MO, USA). Recombinant Human TGF-β was obtained from PeproTech (Rocky Hill, NJ, USA). AS-30D cells were cultured in DMEM with 10% FCS; cells were incubated with 2 ng/ml of TGF-β for 24 or 48 h.
8
Immunohistochemical Analysis of Breast Tissue Microarrays
9
Antibody Detection for EMT Markers
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Primary antibodies against AGO2 (ab186733) and Survivin (ab469) were purchased from Abcam Inc. (Cambridge, UK). Antibodies for detecting Snail (#3895) and Vimentin (#5741) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Primary antibody for GAPDH (sc-25778) and secondary antibodies including anti-rabbit IgG (sc-2004) and anti-mouse IgG (sc-2005) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).
10
Immunocytochemistry for Breast Cancer Markers
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For immunocytochemistry (ICC), 1–2 × 105 cells were seeded onto glass coverslips in 6-well plates in regular media. For assessing PR, cells were treated with 10 nM E2 for 48 h prior to collection. Cells were washed twice with PBS and fixed with ice-cold 70% acetone/30% methanol for 5 min. Fixed cells were blocked with 10% normal goat serum (Vector Labs, Burlingame, CA) in 0.05% TBS-T for 30 min. Primary antibodies were as follows: ERα (SP1, 1:200, Thermo-Fisher, Waltham, MA), PR (1294, 1:50, [26 (link)], CK5 (ab75869, 1:400, Abcam, Cambridge, MA), CK8/18 (NCL-L-5D3, 1:2000, Leica Biosystems), and Vimentin (5741, 1100, Cell Signaling, Danvers, MA) for 1 h. Secondary antibodies were A11029 (green) and/or A11037 (red) (Invitrogen, 1:200) for 30 min followed by counterstaining with 0.1 μg/mL DAPI. Phase contrast images were captured using a Nikon TiE microscope (Nikon, Melville, NY) equipped with a digital camera and NIS Elements 4.6 software. Fluorescent images were captured using an Olympus BX40 microscope equipped with a digital camera and cellSens Standard 1.13 software. Adobe Photoshop CC 2019 was used to perform minimal linear adjustments to brightness/contrast and to assemble pictures into multipanel figures. Immunohistochemistry (IHC) of PDX was performed with the same antibodies for the indicated markers as previously described [21 (link)].
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