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

Developed and validated for fluorescence multiplex IHC — with a published application

Published application: automated multiplex immunofluorescence using DIA-FX3 — Picard et al. (2023)

  • Nuclear FOXP3 · human FFPE tissue
  • FOXP3 × CD112R multiplex documented
  • Six-marker panel using the Opal Polaris 7-colour kit (Picard et al., 2023)

Clone FX3 has been characterized on hundreds of tissues and validated for multiplex fluorescence IHC.

Protocols and published conditions · Publication · Gallery

FX3 is a mouse monoclonal anti-FOXP3 antibody (clone FX3, catalogue number DIA-FX3, ONCOdianova GmbH) for nuclear FOXP3 detection in human FFPE tissue and fluorescence multiplex IHC research. Clone FX3 has been developed and validated for fluorescence multiplex IHC studies of FOXP3 expression in human tissues and detects FOXP3-positive tumour-infiltrating lymphocytes in FFPE human tumours. Documented for brightfield IHC and fluorescence multiplex IHC, with tonsil as positive control and a nuclear staining pattern of FOXP3-positive lymphocytes; DIA-FX3 was used in a peer-reviewed automated multiplex immunofluorescence study.

  • Host / IsotypeMouse · IgG2a/κ
  • ReactivityHuman
  • ApplicationsIHC on FFPE tissue · multiplex fluorescence IHC · Western blot
  • IHC starting range1:100 – 1:200
  • Positive controlTonsil · nuclear
  • Format100 µl, lyophilized
Product image of the ONCOdianova anti-FOXP3 antibody clone FX3: FOXP3 immunohistochemistry of FOXP3-positive regulatory T cells in the stroma of a colorectal adenocarcinoma
Product image · Clone FX3 · DIA-FX3
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

Published application

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

Evidence layer 03

Documented applications and images

Brightfield IHC and fluorescence multiplex IHC on human FFPE tissue, Western blot as listed on the product page; 8 original ONCOdianova figures (tonsil, colorectal, ovarian, pancreatic and stomach carcinoma; FOXP3 × CD112R/PVRIG multiplex), a manual protocol and the published multiplex conditions.

Brightfield and multiplex gallery: section P·03 ↓

Controlled product data.

ProductAnti-FOXP3 (Human) from Mouse, clone FX3 – 100 µl
CloneFX3 (monoclonal)
Catalog numberDIA-FX3
Distributor catalog numberODN-DIA-FX3 (BIOZOL)
Product categoryCancer Immunology IHC marker
TargetFOXP3 — forkhead box protein P3 (scurfin), a transcription factor of regulatory T cells (Gene ID 50943, UniProt Q9BZS1)
ImmunogenRecombinant peptide
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, ONCOdianova gallery, published application) · Western blot (ONCOdianova product page)
IHC starting range1:100 – 1:200 IHC-P (datasheet); 1:100 in the ONCOdianova manual protocol; 1:200 in the published Opal multiplex panel (Picard et al. 2023, 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 ONCOdianova manual protocol)
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
Datasheet designationAnti-FOXP3 / DIA-FX3 — Mouse monoclonal marker Treg cells (Regulatory T cells), Clone FX3
Manufacturer / BrandONCOdianova GmbH
StatusFor Research Use Only. Not for use in diagnostic procedures.

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

IHC protocols and published applications.

ONCOdianova application protocol

Manual FFPE IHC — ONCOdianova protocol

Antibody
DIA-FX3 · Mouse IgG2a/κ · human FOXP3
Reconstitution
restore to 100 µl · sterile distilled water · gentle shaking for 10 minutes
Pretreatment
121 °C · 5 min · pH 7.8
Primary antibody
1:100 · 60 min at 37 °C
Detection
EnVision HRP rabbit/mouse · 30 min at 37 °C

Source: ONCOdianova product page DIA-FX3, tab “IHC protocols” — “Manual stain with autoclave” (header line “Staining protocols for anti-human FOXP3 antibody clone FX3”). The procedure of datasheet DIA-FX3 is kept separate in block A.

External third-party published application

External Published Multiplex Application

Antibody
DIA-FX3 · Dianova
Dilution
1:200
Application
Automated multiplex immunofluorescence
Material
Human FFPE sections
Panel
Six-marker panel using the Opal Polaris 7-colour kit (NEL861001KT, Akoya Biosciences)
Platform
LabSat™ Research Automated Staining Instrument (Lunaphore Technologies SA)
Research context
Pilocytic astrocytoma · multiplex subset of the study: 24 cases

Picard D. et al., Acta Neuropathologica 2023;146:551–564 · doi:10.1007/s00401-023-02626-5 · PMID 37656187 — methods section “Multiplex immunofluorescence”: “FoxP3 (1:200, DIA-FX3, Dianova)”. The dilutions of the other panel markers belong to other antibodies and are listed in block C.

The protocols and published workflows shown here represent documented application examples. The documented conditions provide practical starting points for laboratory establishment of DIA-FX3 workflows across tissue, fixation, platform and detection settings.

Three sources, three application contexts. Block A presents the current datasheet conditions for laboratory establishment. Block B presents the manual ONCOdianova FFPE-IHC protocol. Block C presents the DIA-FX3 multiplex conditions published by Picard et al. 2023. The reported dilutions remain linked to their respective documented application contexts.

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. General recommendation of the product table: 1:100–1:200 (IHC-P)
  3. Biotin/(strept)avidin-based detection possible (e.g. Vectastain® Elite® ABC-HRP kit/AEC)
  4. Polymer-based detection (e.g. Dako EnVision™ detection system, peroxidase/DAB): antibody at 1:100–1:200
  5. Suited for automated staining platforms (datasheet)
  6. Run positive and negative controls in parallel; positive control: tonsil; expected pattern: nuclear
B · ONCOdianova product documentationManual 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 ONCOdianova product information additionally states that the antibody is 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-FX3Picard et al., Acta Neuropathol 2023, Methods “Multiplex immunofluorescence”

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

  1. Human FFPE sections of pilocytic astrocytoma (multiplex subset: 24 cases); six-marker panel using the 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 identifies the antibody as “DIA-FX3, Dianova”.

How to read the staining.

Expected pattern

Nuclear, FOXP3-positive lymphocytes

The datasheet describes a nuclear visualization with tonsil as positive control. The documented ONCOdianova figures illustrate nuclear FOXP3 staining in tumour-infiltrating lymphocytes in colorectal, ovarian, pancreatic and stomach carcinoma and in tonsil.

Documented validation scope

Product validation and documented use

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

Research use

Research interpretation of nuclear FOXP3 staining

FX3 visualizes nuclear FOXP3 protein in human FFPE tissue. In tissue-based research, FOXP3-positive cells can be evaluated together with morphology, tissue compartment and complementary immune markers according to the study design.

FOXP3 in research.

Clone FX3 has been developed for the detection of FOXP3 in routine formalin-fixed, paraffin-embedded tissue specimens (IHC FFPE) and validated for fluorescence multiplex IHC studies of FOXP3 expression in human tissues. Reactivity according to datasheet DIA-FX3 (version 25 Feb 2025/03; identical wording in V02, 22 Feb 2022):

“FOXP3 (Forkhead box protein P3) is mainly expressed in Regulatory T (Treg) cells, a subset of CD4+ T-cells, that play a suppressive role in the immune system. Treg cells ensure immune homeostasis through their ability to suppress the activation and function of leukocytes. FOXP3 has emerged as a prominent target for the development of new immunotherapies for cancer and autoimmune diseases.

The transcription factor FOXP3 is important for the development and inhibitory function of regulatory T-cells (Treg) and acts either as a transcriptional repressor or as a transcriptional activator depending on its interactions with other transcription factors, histone acetylases and deacetylases. FOXP3 coordinates the suppressive activity of Treg cells by activation of different genes, including CTLA4 and TNFRSF18, paralleled by repression of genes encoding cytokines such as interleukin-2 (IL2) and interferon-gamma (IFNG).”

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 FOXP3. Clone-specific application data for FX3 are given in the product data, the protocols, the gallery and the publication section of this page.

  • 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
  • Research evaluationNuclear FOXP3 staining can be evaluated together with morphology, tissue compartment and complementary markers according to the study design
  • 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.

FOXP3 in translational immuno-oncology research.

Target-level

Translational context

FOXP3 is used to study regulatory T-cell populations in the tumor microenvironment. FOXP3-positive populations have been included in tissue-based and multiplex analyses of baseline and on-treatment biopsies from patients receiving checkpoint immunotherapy (PEMBRO-RT trial, multiplex immunofluorescence including FOXP3; van der Woude et al. 2022). Details: FOXP3 in the research overview.

Clone

What the clone can be used to study

Clone FX3 can be used to study FOXP3-positive, nuclear-staining lymphocytes in human FFPE tumors by brightfield and multiplex IHC; it is named in a peer-reviewed study using automated multiplex immunofluorescence, and ONCOdianova documents FOXP3 × CD112R multiplex staining.

Target-level

Translational research context

Published studies provide translational target-level context for FOXP3 research in the tumour microenvironment and in treatment-associated tissue studies.

Clone-specific evidence and target literature.

  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
    Published application: automated multiplex immunofluorescence using DIA-FX3 on human FFPE sections of pilocytic astrocytoma (multiplex subset of 24 cases). Six-marker panel using the Opal Polaris 7-colour kit (tyramide signal amplification): 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

Picard et al. 2023 (PMC10500011) document the published clone-specific application of DIA-FX3.

  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

Entry 7 carries a published correction (2006). Entries 1–8 are the general references of the datasheet; entries 9 and 10 address FOXP3 immunohistochemical methodology. Picard et al. 2023 documents a published clone-specific application of DIA-FX3. The additional literature provides scientific context on FOXP3 biology, regulatory T-cell research and immunohistochemical methodology.

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. Clone FX3 was developed for the detection of FOXP3 in routine formalin-fixed, paraffin-embedded tissue specimens; the datasheet gives a starting range of 1:100 to 1:200 (IHC-P). 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) used DIA-FX3 at 1:200 in an automated six-marker multiplex immunofluorescence panel (Opal Polaris 7-colour kit) on human FFPE sections of pilocytic astrocytoma.

Is FX3 validated for fluorescence multiplex IHC?

Yes. Clone FX3 has been developed and validated for fluorescence multiplex IHC studies of FOXP3 expression in human tissues (ONCOdianova product information and datasheet), and the gallery shows FOXP3 × CD112R/PVRIG multicolor immunofluorescence of normal tonsil. Published application: Picard et al. 2023 used DIA-FX3 in an automated Opal multiplex panel. Every new panel should be verified for tissue, antibody order, retrieval, dilution and detection.

Which dilution is a suitable starting point for FX3?

The datasheet recommends 1:100 to 1:200 (IHC-P) on FFPE sections; the ONCOdianova manual protocol uses 1:100. The published multiplex study used 1:200 within an Opal panel – a study-specific condition. The optimal dilution should 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.

How can FOXP3-positive cells be evaluated in tissue research?

FX3 visualizes nuclear FOXP3 protein in FFPE tissue. Depending on the research question, FOXP3-positive cells can be evaluated together with morphology, tissue compartment and complementary immune markers.

What is the documented validation scope of clone FX3?

Clone FX3 was developed for the detection of FOXP3 in routine FFPE tissue, has been characterized on hundreds of tissues and validated for fluorescence multiplex IHC studies of FOXP3 expression in human tissues; it detects FOXP3-positive tumour-infiltrating lymphocytes in FFPE human tumours (ONCOdianova product information). Published clone-specific application: 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?

Yes. The datasheet states that the antibody is suited for immunohistochemical staining on automated platforms, and the ONCOdianova product information names it applicable for automated staining procedures. Picard et al. 2023 performed their multiplex staining with DIA-FX3 on the LabSat™ automated instrument.

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 ONCOdianova “FOXP3 IHC-Gallery” is presented 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.

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 ONCOdianova product information where indicated. Related: R12 (anti-CD112R/PVRIG) · TG1 (anti-TIGIT) · TG2 (anti-TIGIT) · TC8 (CD8) · KK3 (CD73).

For Research Use Only. Not for use in diagnostic procedures.