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Vu 1d9

Manufactured by Abcam
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VU-1D9 is a primary antibody that recognizes the MAPK1 protein. It is suitable for use in various applications, including Western blotting and immunohistochemistry.

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Lab products found in correlation

Egfr1, by Abcam (2 mentions) Ebiosn3, by Thermo Fisher Scientific (2 mentions) Fcs express 6, by De Novo Software (1 mentions) Cytoflex s flow cytometer, by BD (1 mentions) Epcam pe, by Abcam (1 mentions) E cadherin pe, by BioLegend (1 mentions) Mopc 21, by BD (1 mentions) Axio observerz 1 apotome 2 inverted scope, by Zeiss (1 mentions) Dylight 488, by Abcam (1 mentions) Alexa fluor 647, by BioLegend (1 mentions) Ficoll pacque plus, by GE Healthcare (1 mentions) Magnetic activated cell sorting (macs), by Miltenyi Biotec (1 mentions) Thermomixer comfort, by Eppendorf (1 mentions)

6 protocols using vu 1d9

1

Flow Cytometric Analysis of EMT Markers

Cited 2 times
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Cells were collected, stained, and analyzed by flow cytometry (BD Cytoflex S flow cytometer) as follows. Staining for membrane surface molecules included use of the following antibodies: EpCAM-PE (phycoerythrin): [VU-1D9] (Abcam) and isotype control [MOPC-21] (BD Pharmingen); E-cadherin-PE: CD324 [DECMA-1] BioLegend) and isotype control [A95-1] (BD Pharmingen); and N-cadherin-APC (Allophycocyanin): CD325 [8C11] (BioLegend) and isotype control [27 (link)–35 (link)] (BD Pharmingen). Staining was performed following standard methods. Cells were stained for 30 min (EpCAM) and 20 min (E-cadherin and N-cadherin). For intracellular staining of CCT2 we used the antibody CCT2-PE: [NP_006422] (LSBio) and the isotype control [MOPC-21] (LSBio), following the method in ThermoFisher Scientific’s “Protocol A: two-step protocol: intracellular (cytoplasmic) proteins” and incubating the antibody for 70 min. When optimizing the CCT2 intracellular stain for CSS Autoprep conditions, we adjusted the antibody staining protocol for CCT2-PE to match the 20 min in the CSS Autoprep automated conditions. All data was generated using FCS Express 6 software (De Novo).
Cox A., Martini A., Ghozlan H., Moroose R., Zhu X., Lee E., Khaled A.S., Barr L., Alemany C., Fanaian N., Griffith E., Sause R., Litherland S.A, & Khaled A.R. (2022). Chaperonin containing TCP1 as a marker for identification of circulating tumor cells in blood. PLoS ONE, 17(6), e0264651.
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2

Immunophenotyping of Tumor Cells by FNAB

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A perCP/Cy5.5 conjugated EpCAM mouse anti-human monoclonal antibody (VU-1D9, abcam) and a FITC conjugated CD44 mouse anti-human (clone G44-26, BD pharmingenTM) and their respective isotype controls, mouse IgG1 monoclonal MOPC-21 (perCP/Cy5.5, abcam) and Dnk pAb to Ms IgG (Dylight®488, abcam) were diluted with RPMI media to a working solution 1:200. FNAB tumor samples were loaded into nine separate channels. The antibody solution containing both the EpCAM and CD44 was perfused into three channels. The isotype control antibodies, used to evaluate for nonspecific binding, were perfused into three other channels. The last three channels contained media only to serve as background auto-fluorescence controls for tissue. One channel was not needed and therefore not used for this experiment. A flow rate of 125 μL/hr was used for all channels. Fluorescent imaging was performed by a Zeiss Axio ObserverZ.1/ApoTome.2 inverted scope using a 378HE Green Fluorescent Protein (GFP) filter (excitation: 450-490nm /emission: 500-550nm) and a 45 Texas Red (TR) filter (emission: 540-580/excitation: 593-668nm) over a 24-hour period.
Holton A.B., Sinatra F.L., Kreahling J., Conway A.J., Landis D.A, & Altiok S. (2017). Microfluidic Biopsy Trapping Device for the Real-Time Monitoring of Tumor Microenvironment. PLoS ONE, 12(1), e0169797.
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3

Capturing Metastatic Prostate Cancer CTCs

Cited 1 time
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Whole blood was collected from patients with metastatic prostate cancer. The study was approved by the University of Wisconsin Institutional Review Board and patients supplied written informed consent. PBMCs were isolated using a Ficoll-Pacque Plus (GE Healthcare) gradient before undergoing CD45 depletion (MACS, Miltenyi Biotec). The VERSA 13 (link),37 (link),38 (link) platform was used to capture CTCs with an anti-EpCAM antibody (R&D) and stain for extracellular markers EpCAM [VU-1D9] (Phycoerythrin, Abcam), CD45, CD34, and CD11b (AlexaFluor 647, BioLegend).
Tokar J.J., Stahlfeld C.N., Sperger J.M., Niles D.J., Beebe D.J., Lang J.M, & Warrick J.W. (2020). Pairing microwell arrays with an affordable, semi-automated single-cell aspirator for the interrogation of circulating tumor-cell heterogeneity. SLAS technology, 25(2), 162-176.
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4

Immunohistochemical Analysis of CRC Markers

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Tumoural and non-neoplastic tissue sections from CRC patients were subjected to immunohistochemical staining (Fig. 3a and Supplementary Fig. 24). Primary monoclonal antibodies against EGFR (EGFR1, Abcam, 1:50 dilution), EpCAM (VU-1D9, Abcam, 1:100 dilution), and CD24 (eBioSN3, ebioscience, 1:100 dilution) were used and the staining was conducted on a Ventana BenchMark XT automated slide stainer (Ventana Medical Systems, Inc.), according to the manufacturer’s recommendations. Stained images were scored, based on the fraction of positive cells among total cancer cells, by a pathologist (G.Y.) who was blind to clinicopathological variables and EV profiling results. Positive cells were defined by positively stained cytoplasmic or membranous pattern within cancer cells in the face of concurrent negative labelling in non-neoplastic tissues. The marker expression was ranked from 0 (negative) to 1 (positive) with the increment of 0.1; this level was used as an ordinal variable in Spearman’s rank test with EV profiling results.
Park J., Park J.S., Huang C.H., Jo A., Cook K., Wang R., Lin H.Y., Deun J.V., Li H., Min J., Wang L., Yoon G., Carter B.S., Balaj L., Choi G.S., Castro C.M., Weissleder R, & Lee H. (2021). A high-throughput magneto-electrochemical array for the integrated isolation and profiling of extracellular vesicles from plasma. Nature biomedical engineering, 5(7), 678-689.
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5

Quantification of Spiked Cancer Cells

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Cells from the cancer cell line MFC-7 were cultured in Dulbecco’s modified Eagle medium (DMEM) including 10% fetal calf serum (FCS) at 10% CO2 in a 25 cm2 flask. Cells were washed with warm PBS and detached with trypsin after 2 min incubation and rinsed with warm DMEM. After centrifugation, the cells were re-suspended in warm BSA/PBS 0.1% w/V and counted. 100.000 cells in warm PBS/BSA were then incubated with either 0.5 μg biotinylated Anti-EpCAM (VU-1D9, Abcam, UK) or biotinylated Anti-HER-2 (3B5, Abcam, UK) in a shaker (Eppendorf Thermomixer Comfort, Hamburg, Germany) revolving at 300 rpm at 37 °C for 40 min. The cells are then centrifuged and suspended in warm PBS. The ratio of dead cells is counted before spiking. To achieve a precise number of spiked cancer cells, the suspension is diluted to 10.000 cells/mL; 5 drops of 10 μL suspension are then pipetted on a glass slide and the cells are counted.
Brinkmann F., Hirtz M., Haller A., Gorges T.M., Vellekoop M.J., Riethdorf S., Müller V., Pantel K, & Fuchs H. (2015). A Versatile Microarray Platform for Capturing Rare Cells. Scientific Reports, 5, 15342.
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6

Immunohistochemical Analysis of CRC Markers

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Tumoural and non-neoplastic tissue sections from CRC patients were subjected to immunohistochemical staining (Fig. 3a and Supplementary Fig. 24). Primary monoclonal antibodies against EGFR (EGFR1, Abcam, 1:50 dilution), EpCAM (VU-1D9, Abcam, 1:100 dilution), and CD24 (eBioSN3, ebioscience, 1:100 dilution) were used and the staining was conducted on a Ventana BenchMark XT automated slide stainer (Ventana Medical Systems, Inc.), according to the manufacturer’s recommendations. Stained images were scored, based on the fraction of positive cells among total cancer cells, by a pathologist (G.Y.) who was blind to clinicopathological variables and EV profiling results. Positive cells were defined by positively stained cytoplasmic or membranous pattern within cancer cells in the face of concurrent negative labelling in non-neoplastic tissues. The marker expression was ranked from 0 (negative) to 1 (positive) with the increment of 0.1; this level was used as an ordinal variable in Spearman’s rank test with EV profiling results.
Park J., Park J.S., Huang C.H., Jo A., Cook K., Wang R., Lin H.Y., Deun J.V., Li H., Min J., Wang L., Yoon G., Carter B.S., Balaj L., Choi G.S., Castro C.M., Weissleder R, & Lee H. (2021). A high-throughput magneto-electrochemical array for the integrated isolation and profiling of extracellular vesicles from plasma. Nature biomedical engineering, 5(7), 678-689.
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