On day 18 post-immunization, eyes and cervical draining lymph nodes of EAU mice (each group, n=3) were collected, and cell suspensions were prepared. The cells were surface stained with anti-CD4 antibody (BioLegend) for 30 min at 4°C. After fixation and permeabilization, the cells were stained with an anti-Foxp3 antibody (BioLegend) to detect Foxp3+ cells. For intracellular staining of interferon (IFN) -γ and interleukin (IL)-17, cells were were pretreated for 4 to 6 h with 50 ng/ml photoblog 12-myristate 13w-acetate, 1μg/ml ionomycin, and 1μg/ml brefeldin A (Sigma-Aldrich), and then incubated with antibodies against IFN-γ and IL-17 (BioLegend) after fixation and overnight permeabilization. Data collection was performed on a FACS Calibur flow cytometer (BD Biosciences, USA), and analyzed using flow cytometry software (FlowJo, USA).
Anti foxp3 antibody
Manufactured by BioLegend
Sourced in United States
The Anti-FOXP3 antibody is a reagent used in research applications. It is designed to detect the FOXP3 protein, which is a transcription factor that plays a key role in the development and function of regulatory T cells. The antibody can be used in various immunological techniques, such as flow cytometry, immunohistochemistry, and Western blotting, to identify and study FOXP3-expressing cells.
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Lab products found in correlation
Anti cd4 antibody, by BioLegend (2 mentions)
Anti cd4, by BioLegend (2 mentions)
Antibodies against ifn γ and il 17, by BioLegend (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Canto 2 flow cytometer, by BD (1 mentions)
Anti cd3, by BioLegend (1 mentions)
Anti ccr5, by BioLegend (1 mentions)
Anti cd8, by BioLegend (1 mentions)
Anti α sma antibody, by Agilent Technologies (1 mentions)
Anti cd68 antibody, by Agilent Technologies (1 mentions)
Anti col3a1 antibody, by Bioss Antibodies (1 mentions)
Rabbit anti mouse cd68 antibody, by Bioss Antibodies (1 mentions)
Diva software, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Anti cd4 antibody, by BD (1 mentions)
Foxp3 perm buffer, by BioLegend (1 mentions)
Gb21303, by Wuhan Servicebio Technology (1 mentions)
Ab104139, by Abcam (1 mentions)
Af4012, by Affinity Biosciences (1 mentions)
9 protocols using anti foxp3 antibody
1
Analyzing Regulatory T Cells and Th17 in EAU
2
Flow Cytometry Phenotyping of Immune Cells
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Tumour-infiltrating lymphocytes and PBLs were suspended in flow buffer (PBS containing 2% fetal bovine serum), and incubated with anti-CD3, anti-CD8, anti-CD4, anti-CCR5 and anti-CCR6 antibodies (Biolegend, San Diego, CA, USA) against surface antigens for 30 min at 4 °C in the dark. Following that, samples were rinsed in flow buffer 2 times. Then, cells were fixed, permeabilised and stained with anti-FoxP3 antibody (Biolegend). After staining, cells were washed twice and analysed using a BD CantoII flow cytometer (Becton Dickinson, San Jose, CA, USA). To detect nonspecific signals, concentration- and isotype-matched nonspecific antibodies were used.
3
Quantifying Tumor-Associated Immune Cells
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Paraffin embedded tissues were analyzed using immunohistochemical staining [49 (link)] with the following primary antibodies: anti-CD68 antibody (DAKO, Carpinteria, CA), anti-α-SMA antibody (DAKO, Carpinteria, CA), anti-CCL7 antibody (Gen Way Biotech, San Diego, CA), anti-COL3A1 antibody (Bioss, Beijing, China), anti-FOXP3 antibody (Biolegend, San Diego, CA) and rabbit anti-Mouse CD68 antibody (Bioss, Beijing, China). For quantification of tumor stromal cells within the tumor area, CD68 was used as a pan-macrophage marker, α-SMA was used to detect cancer activated fibroblasts adjacent to RCC cells [50 (link)], FOXP3 was used as a specific marker for regulatory T cells (Tregs) [22 (link)], and mast cells were assessed using the routine toluidine blue staining method [21 (link)]. Each tumor section was evaluated by using 20× objective lens, and five independent areas with the most abundant positive cells were selected, digitally photographed, and manually counted under a microscope. The average positive cell counts for each patient were used for statistical analysis. For quantification of CD68+ cells in CDX xenografts, four sections from each xenograft were randomly selected and quantified as described above, the average positive cells for each mouse were used for statistical analysis. Results were confirmed by two pathologists in a double-blind analysis.
4
Isolation and Analysis of Regulatory T Cells
5
Immunofluorescence Analysis of GSDMD and Immune Cells in Tumor Tissues
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TC-1-GSDMD-NT cells on the slides were collected and treated with 4% paraformaldehyde containing 0.1% Triton X-100 for 10 min, followed by three washes with PBS. Then, the cells were blocked with 2% BSA in PBS for 1 h and incubated with rabbit polyclonal anti-GSDMD antibody (Affinity, AF4012; 1:100 in 2% BSA) for 2 h at RT, followed by three washes with PBS. Next, the samples were incubated with FITC-conjugated secondary antibody (1:2,000 in 1% BSA, Proteintech) for 2 h at RT. After washing five times with PBS, the nuclei were stained with DAPI (ab104139, Abcam) for 10 min. All of the samples were examined by confocal microscopy. Tumor tissues were collected and fixed in formalin, embedded in paraffin, and sectioned. After deparaffinization and hydration, the slides were immersed in EDTA antigen retrieval buffer. Then, the slides were incubated with anti-CD4 antibody (Biolegend) and anti-Foxp3 antibody (Biolegend) or rabbit polyclonal anti-GSDMD antibody (Affinity, AF4012, 1:100) overnight at 4 °C. Next, Cy3-conjugated secondary antibody (Servicebio, GB21303; 1:2,000 in 2% BSA) was added, followed by incubation at room temperature for 1 h. Then, the slides were incubated with DAPI (ab104139, Abcam) at RT for 10 min. All of the sections were examined by fluorescence microscopy.
6
Comprehensive Immune Cell Analysis in Mice
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The spleen and lymph node (LN) of treated mice were collected and suspended as single cells with red blood cell lysis buffer. Colorectal tissues from treated mice were collected and suspended as single cells with collagenase IV. Cells were extracellularly stained with anti‐CD3, anti‐CD4, anti‐CD25, anti‐F4/80, anti‐CD11b, anti‐CD80 and anti‐CD206 antibodies. Subsequently, the cells were fixed and permeabilized with Perm/Fix solution (eBioscience, San Diego, CA, USA) for 30 min. at 4°C. Finally, the cells were intracellularly stained with anti‐Foxp3 antibody (Biolegend, San Diego, CA, USA) at 4°C for 30 min. in the dark. Directly conjugated, isotype‐matched, rat antimouse Abs (Biolegend) served as controls. After washed with PBS twice, the pellet was resuspended in 400 μl PBS and analysed by the Calibur flow cytometer (BD).
7
Quantifying T-cell Subsets in Autoimmune Hepatitis
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PBMCs were isolated from AIH patients and healthy volunteers as mentioned above. In addition, single-cell suspensions were obtained by mechanical disruption of mouse spleens through 70-μm cell strainers. Erythrocytes were lysed using lysis buffer. Then, the freshly collected cells were resuspended in 100 μl of PBS and incubated with anti-CD3, anti-CD4, and anti-CD8a antibodies (BioLegend, San Diego, CA, United States ) at 4°C for 30 min. To detect Treg subpopulations, intracellular staining was performed using a transcription factor buffer set (BioLegend, San Diego, CA, United States ). After staining with anti-CD4 and anti-CD25 antibodies, the cells were fixed, permeabilized, and incubated with an anti-FOXP3 antibody (BioLegend, San Diego, CA, United States ). The frequencies of CD4+ CD25+ FOXP3+ Tregs, CD3+ CD4+ T cells, and CD3+ CD8a+ T cells were examined using a flow cytometer (Beckman Coulter, Brea, CA, United States).
8
Pneumococcal Infection in Mice: Lung Histology
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On day 7 after the last immunization, mice were intranasally challenged with 1 × 108 CFU of pneumococcal strain 19F. Mice were sacrificed and lung tissues were removed at 6, 12, and 24 h post-infection. After fixation in buffered 10% formalin, lungs were sectioned and embedded in paraffin, and 5-µm sections were cut. The sections were stained with hematoxylin and eosin (Sigma-Aldrich) and then examined using a light microscope. The degrees of peribronchial inflammation were graded semi-quantitatively following previously described methods (22 (link)).
For immunohistochemistry, sections were retrieved in citrate buffer for 5 min. After natural cooling, sections were incubated with 3% H2O2 and washed three times with PBS, followed by incubation with an anti-FOXP3 antibody (BioLegend) and Rabbit anti-TGF-β1 polyclonal antibody (OmnimAbs, Alhambra, CA, USA) and treatment with streptavidin horseradish peroxidase chemistry according to standard protocols. The mean IODs (integral optical density) of TGF-β1 expression were measured using Image-Pro Plus (Media Cybernetics, Silver Spring, MD, USA).
For immunohistochemistry, sections were retrieved in citrate buffer for 5 min. After natural cooling, sections were incubated with 3% H2O2 and washed three times with PBS, followed by incubation with an anti-FOXP3 antibody (BioLegend) and Rabbit anti-TGF-β1 polyclonal antibody (OmnimAbs, Alhambra, CA, USA) and treatment with streptavidin horseradish peroxidase chemistry according to standard protocols. The mean IODs (integral optical density) of TGF-β1 expression were measured using Image-Pro Plus (Media Cybernetics, Silver Spring, MD, USA).
9
CyTOF Antibody Labeling for FoxP3
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Antibodies were preconjugated from Fluidigm. Anti-FoxP3 antibody from BioLegend (cat: 3200001) was conjugated with 146Nd using the Maxpar® Antibody Labeling Kit from Fluidigm. DNA intercalator-Ir 191/193, 2000X, was from DVS, Cat #.: 201192B, and cisplatin from Enzo Life Sciences.
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