Rat anti mouse foxp3

To detect IFNε in human colon samples, heat-induced epitope retrieval (HIER) was performed with 10 mmol/L Trisma acetate and 1 mmol/L EDTA buffer, pH 6.0 in a microwave. IFNε was detected using rabbit anti-IFNε (Prestige Antibodies), and background staining was prevented with CAS block (Invitrogen). Biotinylated anti-rabbit immunoglobulin G (Vector Laboratories) was used as secondary antibody. To stain FoxP3-expressing cells in mouse colon sections, HIER was done using a Biocare decloaking chamber (Biocare) in Tris-EDTA Buffer (10 mmol/L Tris Base, 1 mmol/L EDTA solution, 0.05% Tween 20, pH 9.0) at 110°C for 5 minutes under pressure. FoxP3 was detected with rat anti-mouse FoxP3 (eBioscience). Biotinylated anti-rat immunoglobulin G (Vector Laboratories) was used as secondary antibody. This was followed by incubation with VECTASTAIN Elite ABC-HRP (Vector Laboratories). Color development was performed with liquid DAB+ Substrate (Dako), and slides were counterstained with hematoxylin (Invitrogen) and coverslipped with D.P.X. neutral mounting medium.
Slides were scanned at 20× magnification with an Aperio Scanscope AT Turbo (Leica Biosystems), and intensity of staining was analyzed with Aperio ImageScope software (Leica Biosystems).

Immunohistochemistry single marker stains were performed for CD8⁺ cytotoxic T-lymphocytes, FoxP3⁺ regulatory T-lymphocytes, Ly6C⁺ myeloid derived suppressor cells and F4/80⁺ macrophages as described earlier [18] . Rat anti-mouse CD8a clone 4SM15 (e-Bioscience 14–0808–82) was used in a 1:100 dilution. Rat anti-mouse Foxp3 (e-Bioscience 14–5773–82) was used in a 1:100 dilution. Monoclonal Rabbit anti-mouse F4/80 (D2S9Rxp) (Bioké, Cell Signaling 70,076 s) was used in a 1:250 dilution. Rat anti-mouse Ly6C, clone ER-MP20 (ThermoFisherScientific MA1_8189) was used in a 1:200 dilution. Microscopic images were digitalized using the Zeiss Axio Slide Scanner using a x20 objective and ZEN2 software (Zeiss). Qupath was used for digital, manual analysis [19] (link). Per slide, four different region of interest (ROI) containing tumor tissue were selected. Positive cells were counted per ROI and a ratio was made of positively counted cells divided by the ROI in µm².

Absolute and relative numbers of lymphocytes of various subpopulations in peripheral blood were counted using flow cytometry (Beckman Coulter). The following antibodies (eBioscience) were used for immune phenotypic analysis of the main subpopulations of lymphocytes: anti-rat CD3 (for T lymphocytes); anti-rat CD4 (for T helpers); anti-rat CD8a (for cytotoxic T cells); anti-rat CD45R (for B lymphocytes); anti-rat CD25 (for activated T cells); anti-mouse/rat Foxp3 (for regulatory T cells); and anti-rat CD314 (for NK cells). Erythrocytes were lysed with OptiLyse C solution (eBioscience).