The following antibodies were purchased from BioLegend: CD16/32 (clone 93), CD45 (clone 30-F11), CD3 (clone 145-2C11), CD4 (clone GK1.5), CD8α (clone 53-6.7), CD25 (clone PC61), CD62L (clone MEL-14), CD69 (clone H1.2F3), PD-1 (clone 29 F.1A12), CD44 (clone IM7), Granzyme B (clone QA16A02), LAG-3 (clone C9B7W), TIM-3 (clone RMT3-23), TCR γ/δ (clone GL3), NK1.1 (clone PK136), and TNF-α (clone MP6-XT22), LAP (clone TW7-20B9), IL-10 (clone JES5-16E3), and Ki-67 (clone 11F6). FOXP3 (clone FJK-16s) and TCR beta (clone H57-597) were purchased from eBioscience.
Foxp3 clone fjk 16s
Manufactured by Thermo Fisher Scientific
Sourced in United States
FoxP3 (clone FJK-16s) is a lab equipment product offered by Thermo Fisher Scientific. It is a monoclonal antibody that targets the FoxP3 protein, which is a transcription factor involved in the development and function of regulatory T cells. The product is intended for research use only and its core function is to detect and analyze FoxP3 expression in various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Cd45 clone 30 f11, by BioLegend (4 mentions)
Cd4 clone gk1, by Thermo Fisher Scientific (4 mentions)
Ifnγ clone xmg1, by BioLegend (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Cd11c clone n418, by BioLegend (3 mentions)
Cd11b clone m1 70, by BioLegend (3 mentions)
Cd4 clone gk1, by BioLegend (3 mentions)
Tim3 clone rmt3 23, by Thermo Fisher Scientific (3 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (3 mentions)
Golgiplug, by BD (3 mentions)
Ab47425, by Abcam (2 mentions)
Af488 affinipure donkey anti mouse igg, by Jackson ImmunoResearch (2 mentions)
Cy3 affinipure donkey anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
Cd8 clone 4sm15, by Thermo Fisher Scientific (2 mentions)
Ki 67 clone sp6, by Thermo Fisher Scientific (2 mentions)
Af488 donkey anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Af647 goat anti rat igg, by Thermo Fisher Scientific (2 mentions)
Pd l1, by BioLegend (2 mentions)
Ctla 4 clone uc10 4b9, by Thermo Fisher Scientific (2 mentions)
Cd11b, by BioLegend (2 mentions)
31 protocols using foxp3 clone fjk 16s
1
Comprehensive Immune Cell Profiling
2
Immunohistochemical Analysis of FoxP3 in Spinal Cord
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The spine was dissected, immediately embedded into Tissue-Tek® (Sakura Finetek, Zoeterwoude, The Netherlands) and frozen on dry ice. Specimens were stored at −80 °C until further analysis. Seven µm-thick sections of the lumbar region were cut on a cryostat, air-dried and post-fixed in acetone. Sections were blocked with 5 % BSA (PAA) and 3 % mouse serum (Sigma-Aldrich) in PBS for 30 min and then incubated with the primary antibody directed against FoxP3 (clone FJK-16 s; eBioscience) diluted 1:4800 in 1 % BSA solution at 4 °C overnight. The next day, sections were incubated with biotin-conjugated rabbit anti-rat IgG (1:400) (Dako, Hamburg, Germany) in 1 % BSA solution at RT for 40 min followed by Neutravidin-Dylight549 (Thermo Scientific, Waltham, MA, USA) at 1:500 dilution in washing solution (TBS + 0.05 % Tween® 20). Counterstaining of cellular nuclei was performed by incubation with Hoechst 33,342 (1:1000 in washing solution; Thermo Scientific). Sections were analyzed on a Zeiss Axioskop 50 epifluorescence microscope using Carl Zeiss Plan-NEOFLUAR 109/0.30 and 409/1.30 objectives and Carl Zeiss filter sets No. 1 (excitation BP 365/12, emission LP 397) and No. 15 (excitation BP 546/12, emission 590 nm) for detection of fluorescence. Digital images were acquired using a Leica DFC350FX camera and software.
3
Multiplex Immunostaining for Tumor Analysis
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Immunostaining for Ki-67 (Vector), HER2 (AB16901; Abcam), and STAT1 (AB47425; Abcam) was performed as previously described6 (link). Secondary antibodies (AF488 AffiniPure donkey anti-mouse IgG, Cy3 AffiniPure donkey anti-rabbit IgG) were from Jackson ImmunoResearch Laboratories. Images were acquired with a Yokogawa spinning disk confocal on an inverted Nikon Ti fluorescence microscope using MetaMorph image acquisition software, and 3–5 fields were analyzed per tumor. Image analysis was performed using a semi-automated in-house platform (NIH ImageJ).
Immunofluorescence for FoxP3 (clone FJK-16s; eBioscience), CD8 (clone 4SM15; eBioscience), and Ki-67 (clone SP6; Thermo Scientific) was performed as previously described21 (link). Secondary antibodies (AF488 donkey anti-rabbit IgG, AF647 goat anti-rat IgG) were from Life Technologies. Tissues were counterstained with DAPI (Invitrogen). Images were acquired on a Nikon Eclipse Ni microscope using NIS Elements software, and 5–10 fields were analyzed per tumor.
Immunofluorescence for FoxP3 (clone FJK-16s; eBioscience), CD8 (clone 4SM15; eBioscience), and Ki-67 (clone SP6; Thermo Scientific) was performed as previously described21 (link). Secondary antibodies (AF488 donkey anti-rabbit IgG, AF647 goat anti-rat IgG) were from Life Technologies. Tissues were counterstained with DAPI (Invitrogen). Images were acquired on a Nikon Eclipse Ni microscope using NIS Elements software, and 5–10 fields were analyzed per tumor.
4
Immune Profiling of Lung Infiltrates
5
Murine Splenic Immune Cell Profiling
6
Isolation and Analysis of Intestinal Lymphocytes
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Caecum and proximal colon were excised and intra epithelial lymphocytes (IEL) and lamina propria lymphocytes (LILP) were prepared essentially as described [52 (link)] with slight ±modification in the tissue digestion step (digestion medium used was RPMI with 10% Foetal calf serum, 0.1% w/v collagenase type I and Dispase II (both Invitrogen), and tissue was digested for 30min at 37°C). Cell suspensions were blocked with anti-FcγR antibody (clone 24G2; eBioscience) and processed with ebioscience fix/perm buffer as per manufacturer’s instructions before labelling with antibodies specific for CD4 (clone GK1.5; eBioscience), Foxp3 (clone FJK-16s; eBioscience) and T-bet (clone TWAJ; eBioscience). All samples were analysed on a FACS LSRII.
7
Quantifying Tumor-Infiltrating Lymphocytes Using Immunofluorescence
8
Comprehensive Immunological Phenotyping
9
Tumor-Infiltrating Immune Cell Profiling
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Single-cell suspensions of transplanted tumors were obtained by digestion with 1 mg/ml Collagenase I (Sigma) and 1 mg/ml Dispase II (Roche) in RPMI containing 5 % FBS for 45 min at 37°C under shaking condition. The cell suspension was passed through a 70 μm nylon mesh and centrifuged. After red blood cell lysis, Fc receptors were blocked with anti-mouse FcR antibody (clone 93, Biolegend) for 15 min at a 1:100 dilution on ice. Then, cells were stained with anti-CD45 (clone 30-F11, Biolegend), CD11b (clone M1/70, Biolegend), Gr-1 (clone RB6-8C5, Biolegend), F4/80 (clone BM8, Biolegend), PDGFRα (clone APA5; Biolegend), CD206 (clone C068C2, Biolegend), CD11c (clone N418, Biolegend) and MHC II (I-A/I-E, clone M5/114.15.2, Biolegend) antibodies for 30 min at a 1:100 dilution on ice. For analysis of Treg cells in the tumor, cells were stained with anti-CD4 (clone GK1.5, Biolegend, 1:100), Foxp3 (clone FJK-16s, eBioscience, 1:50) antibodies using a Foxp3 staining buffer kit following the instructions (eBioscience). Samples were acquired on a Canto II flow cytometer (BD Biosciences) and the data were analyzed with FlowJo software.
10
Isolation and Analysis of Intestinal Immune Cells
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Spleens and mesenteric lymph nodes (mLNs) were removed from mice and disaggregated through a 100 μm sieve. Small intestines were excised and lamina propria lymphocytes (SILP) were prepared essentially as described [80 (link)] with slight modification in the tissue digestion step (digestion medium used was RPMI with 10% Foetal calf serum, 0.1% w/v collagenase type I and Dispase II (both Invitrogen), and tissue was digested for 30 min at 37°C). Cell suspensions were blocked with anti-FcγR antibody (clone 24G2; eBioscience) before labelling with antibodies specific for CD3 (eBio500A2), CD4 (clone GK1.5; eBioscience), Foxp3 (clone FJK-16s; eBioscience), IL-13 (clone eBiol13A; eBioscience), IFNγ (clone XMG1.2; eBioscience), IL-17(eBio17B7; eBioscience), IL-9 (RM9A4e; Biolegend) or p-Smad 2/3 (Santa Cruz). For intracellular cytokine analysis cells were incubated for 12 hours with 1x Cell stimulation cocktail (plus protein inhibitors) (ebioscience). Cells were then stained with antibodies using the eBioscience Foxp3 permibilization kit according to the manufacturer's instructions. For pSmad2/3 staining, an Alexa Fluor 594-labelled donkey anti-goat secondary antibody was used (Invitrogen). All samples were analysed on a FACS LSRII.
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