To prepare cells for flow cytometry, hiPSC-CF were dissociated using 1X TrypLE select (Thermo Fisher Scientific) for 10 min. Cells were centrifuged (200×g, 5 min, room temperature) and resuspended in cold 1% Paraformaldehyde for fixation (20 min, 4 °C). Cells were diluted 1:10 with 1X PBS and centrifuged (200×g, 5 min, room temperature). Cells were then rinsed once with Flow Buffer (1X DPBS, 1% Bovine Serum Albumin (Thermo Fisher Scientific) and 0.1% Triton X-100 (Sigma-Aldrich)) and stored at 4 °C. Fixed cells were then centrifuged (200×g, 5 min, room temperature) and resuspended in Flow Buffer with Rabbit anti-Vimentin primary antibody (1:200) (abcam). An unstained control group was incubated in only Flow Buffer. Cells were incubated with primary antibody or Flow Buffer overnight at 4 °C. Post-incubation, all groups were rinsed with Flow Buffer, centrifuged (200×g, 5 min, room temperature) and resuspended in Flow Buffer with Goat anti-Rabbit Alexa Fluor 647 secondary antibody (abcam) and incubated for 1 h at room temperature, protected from light. Stained cells were rinsed twice, and flow cytometry analysis was performed using a MACSQuant Analyzer 10 (Miltenyi Biotec) and analysis performed using FlowJo (FlowJo, Ashland, OR, USA).
Rabbit anti vimentin primary antibody
Manufactured by Abcam
Rabbit anti-Vimentin primary antibody is a lab equipment product. It is a polyclonal antibody raised in rabbits that specifically binds to the vimentin protein. Vimentin is a type III intermediate filament protein that is expressed in various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit alexa fluor 647 secondary antibody, by Abcam (1 mentions)
Tryple select, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Macsquant analyzer 10, by Miltenyi Biotec (1 mentions)
Tamoxifen, by AstraZeneca (1 mentions)
Matrigel, by BD (1 mentions)
Female balb c nude mice, by Charles River Laboratories (1 mentions)
2 protocols using rabbit anti vimentin primary antibody
1
Flow Cytometry Fixation and Staining
2
Xenograft Tumor Formation and Immunohistochemistry
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Animal studies were carried out according to the Guideline for Animal Experiments in National Cancer Center and Osaka university, which meet the ethical standards required by the law and the guidelines for use of experimental animals in Japan, and approved by the Committee for Ethics in Animal Experimentation of the National Cancer Center (T15-004) and Osaka university (30-084-003). A 100 mL volume of 1 Â 10 7 cells (ELF3-knockout or wild-type cells) in a 1:1 mixture of Matrigel (354234, BD Biosciences) was subcutaneously injected into 6 weeks old female BALB/c nude mice (Charles River Laboratories). Three months after inoculation, tissue sections were fixed with formalin and embedded in paraffin. Five mice used in each group. A 50 mL volume of 1 Â 10 6 HBDEC2 ELF3À/À EMR ELF3-ERT2 cells in a 1:1 mixture of Matrigel (BD Biosciences) was subcutaneously injected into 7-week-old female BALB/c nude mice (Charles River Laboratories). Mice were housed with drinking water supplemented with or without tamoxifen (AstraZeneca; 0.1 mg/mL in 5% sucrose). Twenty-one days after inoculation, tissue sections were fixed with formalin and embedded in paraffin. Three mice were used in each group. IHC was performed using rabbit anti-vimentin primary antibody (1:200, Abcam) with EnVisionþ System-HRP labeled polymer anti-Rabbit (K4002, Dako-Agilent).
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