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P·01 Products / T-cell phenotype

FX3 — anti-FOXP3 for nuclear detection in brightfield and multiplex IHC.

Clone FX3 · Cat. no. DIA-FX3 · Transcription factor FOXP3, nuclear staining pattern

Scientific USP

Peer-reviewed automated 7-colour multiplex immunofluorescence

Picard 2023 · Acta Neuropathologica · DIA-FX3 named in the methods

  • Nuclear FOXP3 · human FFPE
  • FOXP3 × CD112R multiplex documented
  • Opal Polaris 7-colour panel (study-specific)

Publication — panel and conditions as published in the study.

Mouse monoclonal antibody for studying the transcription factor FOXP3 in human formalin-fixed, paraffin-embedded tissue. Documented for brightfield IHC and fluorescence multiplex IHC, with tonsil as positive control and a nuclear staining pattern of FOXP3-positive lymphocytes. DIA-FX3 is named in the methods of a peer-reviewed automated multiplex immunofluorescence study of pilocytic astrocytoma.

View product dataOrderIHC galleryContact ↗
Evidence layer 01

Clone-specific datasheet

Datasheet DIA-FX3 (version 25 Feb 2025/03; product content identical to V02, 22 Feb 2022) documents identity, immunogen, formulation, reconstitution, storage, FFPE-IHC starting conditions, tonsil control, nuclear pattern and three figures (colorectal adenocarcinoma, ovarian carcinoma, FOXP3 × CD112R multiplex of tonsil).

Evidence layer 02

Clone-specific publication

Picard et al., Acta Neuropathologica 2023: automated six-marker multiplex immunofluorescence on FFPE pilocytic astrocytoma, methods name “FoxP3 (1:200, DIA-FX3, Dianova)” with the Opal Polaris 7 colour kit on the LabSat™ instrument, Vectra Polaris scanning and HALO™ analysis.

Evidence layer 03

Documented applications and images

Brightfield IHC and fluorescence multiplex IHC on human FFPE tissue; 8 original ONCOdianova figures (tonsil, colorectal, ovarian, pancreatic and stomach carcinoma; FOXP3 × CD112R/PVRIG multiplex), a manual protocol and the published multiplex conditions. No comparative or diagnostic claim is made.

Brightfield and multiplex gallery: section P·03 ↓

P·02 Technical data

Controlled product data.

ProductAnti-FOXP3 (Human) from Mouse
CloneFX3 (monoclonal)
Catalog numberDIA-FX3
TargetFOXP3 — forkhead box protein P3 (scurfin), a transcription factor of regulatory T cells (Gene ID 50943, UniProt Q9BZS1)
ImmunogenRecombinant peptide (sequence, region, epitope and recognized isoforms not published)
IsotypeMouse IgG2a/κ
Host speciesMouse
ReactivityHuman
ConjugationUnconjugated
Format100 µl, lyophilized; antibody purified from culture supernatant
ReconstitutionRestore to 100 µl with sterile distilled water; gentle shaking for 10 minutes
FormulationPBS, pH 7.4, 2% BSA, 0.05% sodium azide
ApplicationsIHC on standard FFPE sections (datasheet) · fluorescence multiplex IHC (datasheet reactivity text; ONCOdianova gallery; clone-specific publication)
IHC starting range1:100 – 1:200 for polymer-based detection (datasheet); 1:100 in the manual protocol of the previous product page; 1:200 in the published Opal multiplex panel (study-specific); optimal dilution to be determined by the user for tissue, fixation, platform and detection system
Epitope retrievalHeat-induced epitope retrieval required; autoclave 121 °C, 5 min, Tris-EDTA-citrate pH 7.8 recommended
Primary antibody incubation60 min at 37 °C (datasheet and previous product page)
Positive controlTonsil
Staining patternNuclear (FOXP3-positive lymphocytes)
Automated platformsSuited for automated IHC platforms (datasheet); published multiplex staining on the LabSat™ instrument (Picard et al. 2023)
StorageLyophilized at 2–8 °C; long-term at −20 °C (stable for at least one year); reconstituted at 2–8 °C short term (several weeks); avoid repeated freeze/thaw cycles
Associated antibodiesDIA-R12 (anti-CD112R/PVRIG, clone R12) · DIA-TC8 (anti-CD8, clone TC8) — as listed in the datasheet; each clone carries its own evidence
StatusResearch Use Only

The technical specifications above are documented in datasheet DIA-FX3 (version 25 Feb 2025/03), whose product content is identical to V02 (22 Feb 2022), and on the previous ONCOdianova product page where indicated; gene and protein entries link to their registries. Each laboratory must validate its own conditions.

P·04 Documented protocols

Datasheet procedure, manual protocol and published conditions.

Three sources, three scopes. Block A is the current datasheet and the starting point for in-house establishment. Block B is the manual protocol of the previous ONCOdianova product page. Block C shows the multiplex conditions reported in the clone-specific study – they belong to the respective study panel and are not a general recommendation for use. Dilutions are starting values; each laboratory validates tissue, fixation, platform and detection itself.

A · DatasheetImmunohistochemical staining of standard formalin-fixed paraffin sectionsDatasheet DIA-FX3 · version 25 Feb 2025/03 (identical wording in V02, 22 Feb 2022)

Preparation and epitope retrieval

  1. Deparaffinize and rehydrate according to standard procedures
  2. Heat-induced epitope retrieval (HIER) is required
  3. Recommended: autoclave at 121 °C (5 min) in Tris-EDTA-citrate, pH 7.8 (e.g. TEC buffer)

Primary antibody and detection

  1. Incubate primary antibody for 60 min at 37 °C
  2. Biotin/(strept)avidin-based detection possible (e.g. Vectastain® Elite® ABC-HRP kit/AEC)
  3. Polymer-based detection (e.g. Dako EnVision™ detection system, peroxidase/DAB): antibody at 1:100–1:200
  4. Suited for automated staining platforms (datasheet)
  5. Run positive and negative controls in parallel; positive control: tonsil; expected pattern: nuclear
B · Previous product pageManual stain with autoclaveONCOdianova product page DIA-FX3, section “IHC protocols” (as of 30 Aug 2026)
  1. Reconstitution: restore DIA-FX3 to 100 µl with sterile distilled water, gentle shaking for 10 minutes
  2. Pretreatment buffer: 121 °C / 5 min / pH 7.8
  3. Incubation primary antibody: 60 min / 37 °C, dilution 1:100
  4. EnVision HRP rabbit/mouse: 30 min / 37 °C

The previous page added: “Applicable for automated staining procedures and validated for multicolor immunofluorescence (multiplexed IHC)”. Suitability for automated procedures is also stated in the datasheet; the multiplex application is documented by the ONCOdianova product validation (datasheet, gallery) and by the clone-specific study (block C). The 1:100 dilution is the lower end of the datasheet range of 1:100–1:200.

C · Published study conditionsAutomated multiplex immunofluorescence with DIA-FX3 (study context, not a recommendation for use)Picard et al., Acta Neuropathol 2023, Methods “Multiplex immunofluorescence”

Picard et al. 2023 (pilocytic astrocytoma, FFPE sections)

  1. FFPE sections of pilocytic astrocytoma; Opal Polaris 7 colour kit (NEL861001KT, Akoya Biosciences), tyramide signal amplification
  2. FoxP3: DIA-FX3, Dianova · dilution 1:200
  3. Other panel markers of the study: CD4 (1:50), CD8 (1:150), PD-1 (1:300), Iba-1 (1:450), von Willebrand factor (1:120) – reagents of other manufacturers, see publication
  4. Staining on the LabSat™ Research Automated Staining Instrument (Lunaphore Technologies)
  5. Image acquisition: whole-slide multispectral scans at 0.5 µm/pixel on the Vectra Polaris (MOTiF™, Akoya); analysis with HALO™ (Indica Labs)

The 1:200 dilution applies to the described Opal multiplex protocol and does not replace the datasheet starting range of 1:100–1:200 for brightfield IHC. The publication names “Dianova” as supplier (historical brand name of ONCOdianova).

P·05 Interpretation guide

How to read the staining.

Expected pattern

Nuclear, FOXP3-positive lymphocytes

The datasheet describes a nuclear visualization with tonsil as positive control. The ONCOdianova figures show scattered FOXP3-positive nuclei among tumour-infiltrating lymphocytes in colorectal, ovarian, pancreatic and stomach carcinoma and in the T-cell areas of tonsil, without staining of the tumour cells or epithelium in the depicted fields. These are documented fields, not a general expression map; cytoplasmic or tumour-cell signal would require methodological verification.

Documented validation scope

Product validation and documented use

Clone FX3 was developed for the detection of FOXP3 in routine FFPE tissue, characterized on a large number of tissues (“hundreds of tissues” on the previous ONCOdianova homepage) and validated for fluorescence multiplex IHC studies of FOXP3 expression in human tissues; it detects FOXP3-positive tumour-infiltrating lymphocytes in FFPE human tumours. Picard et al. 2023 independently used DIA-FX3 at 1:200 in a peer-reviewed automated multiplex immunofluorescence panel on FFPE tissue.

Scope of evidence

FOXP3 expression is not suppressive function

FOXP3 IHC demonstrates nuclear FOXP3 protein. Activated human conventional T cells can express FOXP3 transiently without regulatory T-cell development,[7, 9] and different anti-FOXP3 antibodies gave poorly correlated results in FFPE tissue.[10] A FOXP3-positive nucleus therefore identifies a FOXP3-expressing cell, not a proven suppressive regulatory T cell; cell assignment should consider morphology, tissue compartment and, where the research question requires it, further markers. No claim of superiority over other antibodies and no diagnostic, prognostic or therapeutic claim is made. For research use only.

P·06 Target background

FOXP3 in research.

FOXP3 (forkhead box protein P3, scurfin; gene FOXP3 on the X chromosome) is a forkhead-family transcription factor that is essential for the development and suppressive function of regulatory T cells and thereby for immune tolerance.[1] Loss-of-function mutations of FOXP3 cause the immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX).[8]

Foxp3 binds and regulates a set of target genes and acts as a transcriptional activator or repressor depending on its interaction partners;[5, 6] the datasheet reactivity text names the activation of CTLA4 and TNFRSF18 and the repression of the cytokine genes IL2 and IFNG. Regulatory T-cell lineage specification involves Foxp3 together with a pre-existing enhancer landscape and epigenetic changes.[2, 3, 4] FOXP3-expressing regulatory T cells are studied in cancer and autoimmunity research; in tissue studies, nuclear FOXP3 staining is used to locate FOXP3-positive cell populations within the tissue architecture.

These statements describe the target. They are not evidence for the analytical performance of clone FX3 and do not establish which cell populations FX3 detects in a given specimen. Clone FX3 is a research tool for tissue-based FOXP3 studies; it carries no diagnostic or therapeutic claim.

  • GeneFOXP3 — NCBI Gene 50943
  • ProteinForkhead box protein P3 / scurfin — UniProt Q9BZS1
  • AliasesFOXP3 · scurfin · IPEX · JM2
  • LocalizationNuclear transcription factor — the basis of the nuclear staining pattern
  • Interpretation limitFOXP3 expression without regulatory T-cell development in activated human T cells — Gavin et al. 2006 [7]
  • Related clonesFOXP3 × CD112R/PVRIG multiplex documented with R12; see also the ONCOdianova anti-TIGIT clones TG1 and TG2

Bracketed numbers refer to the target-literature list below. Target biology is kept separate from clone-specific product claims.

P·07 Publications

Clone-specific evidence and target literature.

Clone-specific evidence (DIA-FX3 named in the source text)

  1. Picard D, Felsberg J, Langini M, Stachura P, Qin N, Macas J, Reiss Y, Bartl J, Selt F, Sigaud R, Meyer FD, Stefanski A, Stühler K, Roque L, Roque R, Pandyra AA, Brozou T, Knobbe-Thomsen C, Plate KH, Roesch A, Milde T, Reifenberger G, Leprivier G, Faria CC, Remke M. Integrative multi-omics reveals two biologically distinct groups of pilocytic astrocytoma. Acta Neuropathologica (2023); 146(4):551–564. doi:10.1007/s00401-023-02626-5 · PMID 37656187 · PMC10500011
    Automated multiplex immunofluorescence (Opal Polaris 7 colour kit, tyramide signal amplification) on FFPE sections of pilocytic astrocytoma; panel CD4, CD8, PD-1, FoxP3 (1:200, DIA-FX3, Dianova), Iba-1 and von Willebrand factor on the LabSat™ instrument, whole-slide multispectral scanning on the Vectra Polaris, analysis with HALO™.    Peer-reviewed article · DIA-FX3 named in the methods

The previous ONCOdianova product page stated that “scientific publications for anti-FOXP3 clone FX3 have been submitted and reference data will be available soon”. The publication above is the clone-specific peer-reviewed use identified and verified in the full text as of 30 Aug 2026.

Target literature cited in the datasheet and method literature

  1. Lu L, Barbi J, Pan F. The regulation of immune tolerance by FOXP3. Nature Reviews Immunology (2017); 17:703–717. doi:10.1038/nri.2017.75 · PMID 28757603Target biology · review
  2. Samstein RM et al. Foxp3 exploits a pre-existent enhancer landscape for regulatory T cell lineage specification. Cell (2012); 151:153–166. doi:10.1016/j.cell.2012.06.053 · PMID 23021222Target biology
  3. Fu W et al. A multiply redundant genetic switch ‘locks in’ the transcriptional signature of regulatory T cells. Nature Immunology (2012); 13:972–980. doi:10.1038/ni.2420 · PMID 22961053Target biology
  4. Ohkura N et al. T cell receptor stimulation-induced epigenetic changes and Foxp3 expression are independent and complementary events required for Treg cell development. Immunity (2012); 37:785–799. doi:10.1016/j.immuni.2012.09.010 · PMID 23123060Target biology
  5. Marson A et al. Foxp3 occupancy and regulation of key target genes during T-cell stimulation. Nature (2007); 445:931–935. doi:10.1038/nature05478 · PMID 17237765Target biology
  6. Zheng Y et al. Genome-wide analysis of Foxp3 target genes in developing and mature regulatory T cells. Nature (2007); 445:936–940. doi:10.1038/nature05563 · PMID 17237761Target biology
  7. Gavin MA et al. Single-cell analysis of normal and FOXP3-mutant human T cells: FOXP3 expression without regulatory T cell development. Proceedings of the National Academy of Sciences USA (2006); 103:6659–6664. doi:10.1073/pnas.0509484103 · PMID 16617117
    Correction published 2006, doi:10.1073/pnas.0603222103    Target biology
  8. Bennett CL et al. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nature Genetics (2001); 27:20–21. doi:10.1038/83713 · PMID 11137993Target biology
  9. Roncador G et al. Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level. European Journal of Immunology (2005); 35:1681–1691. doi:10.1002/eji.200526189 · PMID 15902688FOXP3 antibody methodology
  10. Woo YL et al. FOXP3 immunohistochemistry on formalin-fixed paraffin-embedded tissue: poor correlation between different antibodies. Journal of Clinical Pathology (2008); 61:969–971. doi:10.1136/jcp.2008.056200 · PMID 18413654FOXP3 antibody methodology

Titles quoted verbatim; PubMed status and Crossref metadata (including retraction, correction and expression-of-concern notices) checked on 30 Aug 2026 for every entry: no retraction and no expression of concern; entry 7 carries a published correction (2006), which is noted. Entries 1–8 are the general references of the datasheet; entries 9 and 10 characterize other FOXP3 antibodies and the variability of FOXP3 IHC.

P·08 FAQ

Frequent scientific questions.

What does anti-FOXP3 clone FX3 detect?

FX3 is a mouse monoclonal antibody (IgG2a/κ) raised against a recombinant peptide of human FOXP3 (forkhead box protein P3; gene FOXP3). It is offered as a research reagent for immunohistochemistry on human formalin-fixed, paraffin-embedded (FFPE) tissue, in brightfield IHC and in fluorescence multiplex IHC, with a nuclear staining pattern.

Is FX3 documented for FFPE immunohistochemistry?

Yes. The datasheet states that clone FX3 was developed for the detection of FOXP3 in routine formalin-fixed, paraffin-embedded tissue specimens and gives a starting range of 1:100 to 1:200 for polymer-based detection. ONCOdianova figures show nuclear FOXP3 staining in tonsil, colorectal adenocarcinoma, high-grade serous ovarian carcinoma and further solid cancers.

Are there peer-reviewed publications using clone FX3?

Yes. Picard et al., Acta Neuropathologica 2023 (PMID 37656187) name DIA-FX3 in their methods and used the antibody at 1:200 in an automated six-marker multiplex immunofluorescence panel on FFPE sections of pilocytic astrocytoma. The clone attribution was verified in the full text on 30 August 2026.

Is FX3 validated for fluorescence multiplex IHC?

Yes. The datasheet and the previous product page state that clone FX3 was developed and validated for fluorescence multiplex IHC studies of FOXP3 expression in human tissues, and the gallery shows FOXP3 × CD112R/PVRIG multicolor immunofluorescence of normal tonsil. Independently published evidence includes Picard et al. 2023, who used DIA-FX3 in an automated Opal multiplex panel. Every new panel must be verified for tissue, antibody order, retrieval, dilution and detection.

Which dilution is a suitable starting point for FX3?

The datasheet states 1:100 to 1:200 for polymer-based peroxidase/DAB detection on FFPE sections; the previous product page used 1:100 in its manual protocol. The published multiplex study used 1:200 within an Opal panel – a study-specific condition. The optimal dilution must be determined by the user for each tissue, fixation, platform and detection system.

Which epitope retrieval does FX3 require?

Heat-induced epitope retrieval is required. The datasheet recommends an autoclave at 121 °C for 5 minutes in Tris-EDTA-citrate buffer, pH 7.8 (TEC buffer).

Which positive control and staining pattern are documented for FX3?

Human tonsil, with a nuclear staining pattern of FOXP3-positive lymphocytes. The datasheet names tonsil as the positive-control tissue and “nuclear” as visualization; the gallery includes a tonsil overview and FOXP3 × CD112R/PVRIG multiplex images of normal tonsil.

Does every FOXP3-positive cell represent a functional regulatory T cell?

No. FOXP3 IHC demonstrates nuclear FOXP3 protein expression. In human T cells FOXP3 can be expressed transiently after activation without regulatory T-cell development (Gavin et al. 2006; Roncador et al. 2005), and different anti-FOXP3 antibodies gave poorly correlated results in FFPE tissue (Woo et al. 2008). Interpretation should consider nuclear localization, morphology, tissue compartment and, depending on the research question, additional markers such as CD3, CD4 or CD25. FOXP3 IHC does not measure suppressive function.

What is the documented validation scope of clone FX3?

Clone FX3 was developed for the detection of FOXP3 in routine FFPE tissue, characterized on a large number of tissues (“hundreds of tissues” on the previous ONCOdianova homepage) and validated for fluorescence multiplex IHC of FOXP3 in human tissues; it detects FOXP3-positive tumour-infiltrating lymphocytes in FFPE human tumours. Independently published clone-specific evidence includes automated multiplex immunofluorescence on human FFPE tissue with DIA-FX3 at 1:200 (Picard et al. 2023).

Can FX3 be used on automated staining platforms?

The datasheet states that the antibody is suited for immunohistochemical staining on automated platforms, and the previous product page stated that the protocol is applicable to automated staining procedures. Picard et al. 2023 performed their multiplex staining with DIA-FX3 on the LabSat™ automated instrument.

Is FX3 intended for diagnostic use?

No. FX3 is intended for research use only and is not intended for diagnostic or therapeutic procedures.

P·09 Documents

Data package.

Datasheet

DIA-FX3

Product datasheet, version 25 Feb 2025/03 (product content identical to V02, 22 Feb 2022; updated distribution address). Identity, immunogen, formulation, reconstitution, storage, instructions for use, three figures and eight general references.

Download PDF ↓

PDF · version 2025/03 · 235 KB

IHC gallery

FOXP3 IHC gallery

The “FOXP3 IHC-Gallery” of the previous website is integrated in section P·03 of this page with 8 figures and their legends.

Go to gallery ↓

8 figures · brightfield and multiplex

Safety

MSDS

Material safety data sheet (ONCOdianova, all lyophilized antibodies).

Download PDF ↓

PDF · V02 · 216 KB

Ordering. ONCOdianova products are purchased through our distribution partner BIOZOL Diagnostica Vertrieb GmbH, Oehleckerring 11–13, 22419 Hamburg, Germany — order requests by e-mail: order@biozol.de (CC info@oncodianova.com). See also order information and distributors (in preparation).

The technical data on this page are taken from datasheet DIA-FX3 (version 25 Feb 2025/03; product content identical to V02, 22 Feb 2022) and from the previous ONCOdianova product page where indicated. Related: R12 (anti-CD112R/PVRIG) · TG1 (anti-TIGIT) · TG2 (anti-TIGIT) · TC8 (CD8) · KK3 (CD73).

For Research Use Only. Not for diagnostic or therapeutic use.