Briefly, tissues (tongue, cervical lymph nodes, and spleen) were harvested, fixed, and paraffin embedded. Slides were stained for CK5 (Fitzgerald, 20R-CP003) (1:500) and CD8 (abcam, ab22378) (1:400) antibodies. Quantification of immune infiltration was done using QuPath, an open source software for digital pathology image analysis60 (link). For the quantification, at least three regions of interest (ROI) were selected for each condition and the percentage of positive cells for the CD8 marker was calculated. In order to quantify the immune-fluorescent-stained Foxp3 and CD8 positive cells in the Foxp3DTR mice, we quantified the number of positive cells in each ROI. CD8 antibody (catalog # ab22378) (1:400) was purchased from Abcam (Cambridge, United Kingdom) and FoxP3 antibody (catalog #D608R) (1:200) was purchased from Cell Signaling Technology (Danvers, MA).
Foxp3 antibody
Manufactured by Cell Signaling Technology
Sourced in United Kingdom, Canada
The FOXP3 antibody is a laboratory reagent designed for the detection and analysis of the FOXP3 protein, a transcription factor essential for the development and function of regulatory T cells. This antibody can be used in various immunoassay techniques, such as Western blotting, immunohistochemistry, and flow cytometry, to study the expression and localization of FOXP3 in biological samples.
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Lab products found in correlation
Cd8 antibody, by Abcam (1 mentions)
Ab22378, by Abcam (1 mentions)
20r cp003, by Abcam (1 mentions)
Ventana benchmark xt system, by Roche (1 mentions)
Cell conditioning 1, by Roche (1 mentions)
Dako autostainer link 48, by Agilent Technologies (1 mentions)
F4 80 antibody, by Cell Signaling Technology (1 mentions)
Mpo antibody, by Thermo Fisher Scientific (1 mentions)
Cd3 antibody, by Agilent Technologies (1 mentions)
Immunohistochemistry kit, by Wuhan Servicebio Technology (1 mentions)
Cd8 antibody, by Cell Signaling Technology (1 mentions)
Cd4 antibody, by Abcam (1 mentions)
Cd3 antibody, by Abcam (1 mentions)
Setdb1 antibody, by Abcam (1 mentions)
Interleukin 2 (il 2), by Immunotools (1 mentions)
5 protocols using foxp3 antibody
1
Quantification of Immune Infiltration in Mouse Tissues
2
FOXP3 and CD163 Immunohistochemistry Assay
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From 70 formalin-fixed, paraffin-embedded tissue samples, which were analyzed previously by RNA-Seq and WGS in their frozen tissues [17] (link), five 4-μm-thick sections were serially cut and mounted on pre-coated slides. A FOXP3 assay was then performed using the Ventana Benchmark XT system (Roche). For antigen retrieval, Cell Conditioning 1 (Roche) was poured onto the sections, which were then heated on a slide heater at 95 °C for 64 min. The tissue sections were incubated with × 200 dilution of FOXP3 antibody (Cell Signaling Technology). A CD163 assay was performed using DAKO Autostainer Link 48 (Agilent Technologies). For antigen retrieval, the sections were heated at 97 °C for 20 min with Target Retrieval Solution, High pH (Agilent Technologies). The tissue sections were incubated with × 200 dilution of CD163 antibody (Novocastra, Leica Biosystems).
3
Gastric Immune Cell Quantification
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Paraffin-embedded gastric tissues from five control mice, four C. acnes-colonized mice, and eight mice from H. pylori and coinfected groups were stained using F4/80 antibody (1:100, Cell Signaling Technology, Danvers, MA) to detect macrophages, CD3 antibody (1:400, Agilent Dako, Santa Clara, CA) to detect T cells, FOXP3 antibody (1:100, Cell Signaling Technology, Danvers, MA) to detect Treg cells, CD45 B220 antibody (1:400, Thermo Scientific, Waltham, MA) to detect B cells, and MPO antibody (1:50, Thermo Scientific, Waltham, MA) to detect neutrophils by immunohistochemistry as previously described (5 (link)). Positive cells as a percentage of total cells within the gastric mucosa of whole digital slides were quantified using QuPath imaging software.
4
Immunohistochemical Analysis of Immune Cell Markers
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Tissues were fixed overnight in 4% paraformaldehyde and embedded in paraffin. All IHC staining was performed on 4-µm sections. After deparaffinization, rehydration, antigen unmasking, and endogenous peroxidase blocking, sections were blocked in 5% BSA with 0.1% Triton X-100 in PBS. Sections were incubated overnight at 4°C with primary antibodies. The details of antibodies are as follows: CD3 antibody (Abcam; cat #ab5690; 1:200); CD4 antibody (Abcam; cat #ab183685; 1:200); CD8 antibody (Cell Signaling Technology; cat #98941; 1:200); Foxp3 antibody (Cell Signaling Technology; cat #12653; 1:200); SETDB1 antibody (Abcam; cat #ab12317; 1:200); and FOXM1 antibody (Abcam; cat #ab232649; 1:200). Tissue sections were then stained using an Immunohistochemistry kit (Servicebio; cat #G1215; cat #G1215-200T; cat #G1216-200T). Tissues were observed under a microscope and photographed after being counterstained with hematoxylin, dried, and mounted. For assessment of CD3+, CD4+, CD8+, and Foxp3+ T cell infiltration, positively stained cells were counted from five high-power random fields of each sample, and the numbers averaged for each field. The IHC scores for SETDB1 and FOXM1 staining are assigned using a semi-quantitative five-category grading system as previously described (Sun et al., 2018 (link)).
5
IL-2 Induced STAT5 Phosphorylation in Treg Cells
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Splenic cells were incubated for 10 min at 37°C with increasing doses of IL-2 (0, 0.1, 1, 10 ng/ml) (Immuno Tools) and immediately fixed in 2% paraformaldehyde. Cells were made permeable by incubation in 90% methanol and then were stained with CD4, TCRβ, FOXP3 antibody and primary rabbit antibody to phosphorylated STAT5 or isotype control (Cell Signaling) revealed with an anti-rabbit Alexa-647 conjugated secondary antibody. Stat5 phosphorylation was then determined by flow cytometry in CD4+TCRβ+FOXP3+ cells.
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