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Foxp3 biotin

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Foxp3-Biotin is a biotin-conjugated antibody that specifically binds to the Forkhead box protein P3 (Foxp3), a transcription factor expressed in regulatory T cells. It is used for the identification and quantification of Foxp3-positive cells in flow cytometric analysis.

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Lab products found in correlation

Cm1860 cryostat, by Leica (2 mentions) Dmi8 microscope, by Leica (2 mentions) Streptavidin alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Bovine serum albumin (bsa), by Merck Group (2 mentions) Fluoroshield with dapi, by Merck Group (2 mentions) Laminin alexa fluor 647, by Novus Biologicals (2 mentions) Tris buffered saline (tbs), by Boston BioProducts (2 mentions) Cd11b pe cy5, by Thermo Fisher Scientific (1 mentions) Cd4 pe cy5, by Thermo Fisher Scientific (1 mentions) Fitc conjugated streptavidin, by Thermo Fisher Scientific (1 mentions) Cd8 pe, by Thermo Fisher Scientific (1 mentions) Cd4 pe, by Thermo Fisher Scientific (1 mentions) Il 4 pe, by Thermo Fisher Scientific (1 mentions) Cd11c pe, by Thermo Fisher Scientific (1 mentions) Il 17 pe, by Thermo Fisher Scientific (1 mentions) F4 80 fitc, by Thermo Fisher Scientific (1 mentions) Ifn γ fitc, by Thermo Fisher Scientific (1 mentions) Cd86 fitc, by BD (1 mentions) Ly6g pe, by BD (1 mentions) Gr1 apc, by Thermo Fisher Scientific (1 mentions)

4 protocols using foxp3 biotin

1

FACS Analysis of Myeloid-Derived Suppressor Cells

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Cells were stained with specific antibodies or control isotypes using conventional protocols. The following anti-mouse antibodies were used for FACS analysis of MDSCs: CD11b/PECy5, Gr1/PE, Gr1/APC, CD62L/FITC, F4/80/FITC, CD11c/PE, and MHC CI OVA peptide/Biotin from eBiosciences (San Diego, CA); as well as CD124/PE, CD40/FITC, CD86/FITC, IA-IE/FITC, Ly6C/FITC and Ly6G/PE that were purchased from BD Biosciences (San Jose, CA). Lymphocytes characterization was performed with the next specific antibodies: CD8/PE and CD8/Biotin, CD69/PECy5, CD62L/FITC, CD247/PE, CD4/PE, CD4/PECy5, CD25/PECy5, Foxp3/Biotin, IFN-γ/FITC, IL-4/PE and IL-17/PE, all from eBiosciences. FITC-conjugated Streptavidin was purchased also from eBiosciences. In all cases, cells were previously incubated with anti-mouse FcγR 2.4G2 ascites (HB-197; ATCC) to reduce the non-specific binding. For intracellular staining, cells were fixed and permeabilized, according to the manufacturer’s protocol, with either Foxp3 Staining Buffer Set or IC Fixation and 10X Permeabilization Buffers from eBiosciences. Cells were acquired using both FACScan (BD Biosciences) and Gallios (Beckman Coulter, Miami, FL) flow cytometers and analyzed with Kaluza 1.2 (Beckman Coulter) and FlowJo 5.7.2 (Tree Star Inc., USA) softwares.
Fernández A., Oliver L., Alvarez R., Hernández A., Raymond J., Fernández L.E, & Mesa C. (2014). Very small size proteoliposomes abrogate cross-presentation of tumor antigens by myeloid-derived suppressor cells and induce their differentiation to dendritic cells. Journal for Immunotherapy of Cancer, 2, 5.
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2

Immunofluorescence Staining of Tumor Sections

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After 14 days tumor engraftment, tumors from WT and KO mice were flash frozen in OCT (Fisher) and sectioned into 8μm slices using a CM1860 cryostat (Leica, Wetzlar, Germany). Sections were fixed in pre-chilled acetone then stained overnight at 4°C with 1:100 Foxp3-Biotin (Ebioscience) and Laminin-Alexa Fluor 647 1:500 (Novus, Littleton, CO) in antibody staining buffer - 0.5% Triton X-100 and 1% BSA (Sigma) in TBS (Boston Bioproducts, Ashland, MA). The following day, Streptavidin Alexa Fluor-488 (Fisher) was added for 2 hours at RT, washed, and mounted using Fluoroshield with DAPI (Sigma). Images were taken with a Leica DMi8 microscope with a 20X objective. Data was processed and quantified using imageJ.
Miska J., Lee-Chang C., Rashidi A., Muroski M.E., Chang A.L., Lopez-Rosas A., Zhang P., Panek W.K., Cordero A., Han Y., Ahmed A.U., Chandel N.S, & Lesniak M.S. (2019). HIF-1α Is a Metabolic Switch between Glycolytic-Driven Migration and Oxidative Phosphorylation-Driven Immunosuppression of Tregs in Glioblastoma. Cell reports, 27(1), 226-237.e4.
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3

Immunofluorescence Staining of Tumor Sections

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After 14 days tumor engraftment, tumors from WT and KO mice were flash frozen in OCT (Fisher) and sectioned into 8μm slices using a CM1860 cryostat (Leica, Wetzlar, Germany). Sections were fixed in pre-chilled acetone then stained overnight at 4°C with 1:100 Foxp3-Biotin (Ebioscience) and Laminin-Alexa Fluor 647 1:500 (Novus, Littleton, CO) in antibody staining buffer - 0.5% Triton X-100 and 1% BSA (Sigma) in TBS (Boston Bioproducts, Ashland, MA). The following day, Streptavidin Alexa Fluor-488 (Fisher) was added for 2 hours at RT, washed, and mounted using Fluoroshield with DAPI (Sigma). Images were taken with a Leica DMi8 microscope with a 20X objective. Data was processed and quantified using imageJ.
Miska J., Lee-Chang C., Rashidi A., Muroski M.E., Chang A.L., Lopez-Rosas A., Zhang P., Panek W.K., Cordero A., Han Y., Ahmed A.U., Chandel N.S, & Lesniak M.S. (2019). HIF-1α Is a Metabolic Switch between Glycolytic-Driven Migration and Oxidative Phosphorylation-Driven Immunosuppression of Tregs in Glioblastoma. Cell reports, 27(1), 226-237.e4.
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4

Immunohistochemical Analysis of Retinal Immune Cells

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For arginase-1 staining, mice were perfused transcardially with ice-cold PBS containing 4 U/mL heparin, then with 4% paraformaldehyde. Eyes were enucleated, and frozen on dry ice in OCT. Ten μm sections were cut on a Lyca cryostat and mounted on gelatin coated slides. Sections were then stained for arginase-1 (Santa Cruz Biotechnology, clone V20), CD68 (Biolegend, clone FA11), Iba1 (Biocare Medical, polyclonal) and GFP (Abcam, polyclonal). For CD4 and CD11b staining, mice were perfused transcardially with ice-cold PBS containing 4 U/mL heparin. Eyes were enucleated and frozen on dry ice in OCT. Ten μm sections were cut on a Lyca cryostat and mounted on gelatin coated slides. Slides were post-fixed in 3:1 acetone:ethanol at 4 degrees before staining with the flowing antibodies: CD4-FITC (eBioscience, clone GK-1.5), CD11b (Biolegend, clone M1/70), Foxp3-biotin (eBioscience, clone FKJ-16s). For Foxp3 and CD4 co-staining, CD4 was detected with an anti-fluorescein secondary antibody (Life Technologies) and FoxP3 was detected with Alexfluor 594 conjugated streptavidin (Jackson Immunochemical)
Walsh J.T., Zheng J., Smirnov I., Lorenz U., Tung K, & Kipnis J. (2014). Regulatory T cells in central nervous system injury: A double-edged sword. Journal of immunology (Baltimore, Md. : 1950), 193(10), 5013-5022.
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