Anti foxp3 clone fjk 16s

Co-immunoprecipitation was performed using nuclear complex co-IP kit (Active Motif), according to manufacturer's instructions. 5 × 10⁶ cells were washed with PBS containing phosphatase inhibitors and lysed in hypotonic buffer. Nuclei were isolated by centrifugation and digested using enzymatic shearing cocktail. Digested nuclear lysate was incubated with polyclonal affinity purified rabbit Foxp3 antibody^{9 (link)} overnight at 4 °C. Foxp3 immune-complexes were captured by addition of protein-A conjugated magnetic beads, thoroughly washed, and resuspended in Laemmli sample buffer for SDS-PAGE fractionation and immunoblot analysis. The following antibodies were used for immunoblotting: anti-EZH2 (BD Biosciences Cat# 612666), anti-Foxp3 (clone FJK-16s, eBioscience Cat# 14-57773-82) and anti-histone H3 (Abcam ab1791).

Immunohistochemistry was performed on tumors from 4 mice per treatment condition to quantify FoxP3⁺ tumor infiltrate. Tumors were harvested on day 6 after delivery of 12 Gy or sham RT to the primary tumor in mice bearing a primary or a primary and untreated secondary B78 melanoma tumor. Fresh tumor samples were dissected, cryo-embedded in OCT solution, and sectioned. Frozen sections were fixed in -20°C acetone for 20 minutes, blocked in 5% normal rabbit serum, and labeled overnight at 4°C using a 1:1000 dilution of anti-FoxP3 (clone FJK-16s, eBioscience), anti-CD8 (clone 53-6.7, eBioscience), or non-specific isotype control antibody in 5% rabbit serum PBS with 0.01% Triton x-100. FoxP3 is used as a lineage marker for Tregs, recognizing that this population is functionally heterogeneous and may include rare subsets of other cell types (37 (link)). Labeled cells were detected using the ImmPRESS™ peroxidase secondary and DAB substrate kits from Vector Laboratories. Slides were counterstained with hematoxylin. Three representative images were captured from the cortex of each tumor specimen at 200× magnification using an Olympus BX41 inverted microscope equipped with an Olympus XM10 digital camera. Images were viewed using CellSen Standard software and FoxP3⁺ and CD8⁺ cells were quantified by an individual blinded to the treatment conditions.

For analyses of immune cells by flow cytometry, splenocytes and paraaortic lymph node cells were isolated and stained in PBS containing 2% fetal calf serum. Flow cytometry analysis was performed using BD LSRFortessa (BD Biosciences, San Jose, CA) or Attune NxT Flow Cytometer (Thermo Fisher Scientific, Waltham, MA) with FlowJo software (Tree Star, Ashland, OR). The antibodies used were as follows; anti-TCRβ (clone H57–597; Biolegend, San Diego, CA), anti-CD4 (cloneRM4–5; Biolegend), anti-Foxp3 (clone FJK-16s; eBioscience). Intracellular staining of Foxp3 was performed using the Foxp3 staining buffer set (eBioscience) according to the manufacturer’s instructions. The fluorescent staining was performed after blocking the Fc receptor with the anti-CD16/CD32 antibody. Surface staining was performed according to standard procedures at a density of 1 × 10⁶ cells per 50 μL, and the volumes were scaled up accordingly.

The following antibodies were from Biolegend: anti-CD4 (clone GK1.5); CD8α (clone 53-6.7); CD44 (clone IM7); CD25 (clone PC61); CD45.1 (clone A20); CD45.2 (clone 104); Thy1.1 (clone OX-7); Thy1.2 (clone 30-H12); H-2K^b (clone AF6-88.5); H-2K^d (clone SF1-1.1); IFNγ (clone XMG1.2); IL-17A (clone TC11-18H10.1); pan-CD43 (clone S11), core-2 O-glycosylation CD43 (clone 1B11); KLRG1 (clone 2F1); CD127/IL7Rα (clone A7R34). Anti-FoxP3 (clone FJK-16s) was from eBioscience. Anti-Vβ13 (clone MR12-3) was from BD Biosciences. SIINFEKL peptide loading on H-2K^b MHC and conjugation to PE was done as previously described (43 (link)). Non-viable cells were excluded from analysis with Zombie Aqua Fixable Viability Dye (Biolegend), or DAPI (Sigma-Aldrich). For intranuclear staining, FoxP3/Transcription Factor Staining Buffer (eBioscience) was used per manufacturer’s protocol. Assessment of intracellular T cell cytokine production after short ex vivo stimulus was previously described (10 (link)). Flow cytometric analysis was performed using a 4-laser Fortessa (BD). Sorting of T cells was performed using a 4-laser FACSAria II/III (BD). Alloreactive 4C T cells were defined as Thy1.1⁺ CD4⁺. Naïve OT-I CD8⁺ T cells (CD8⁺CD44^lo) and day 3 OT-I effector CD8⁺ T cells (CD8⁺CD45.2⁺CD44^hi) were sorted on a BD FACSARIA II.