Overview

  • Product name
    Anti-FOXP3 antibody [236A/E7]
    See all FOXP3 primary antibodies
  • Description
    Mouse monoclonal [236A/E7] to FOXP3
  • Host species
    Mouse
  • Specificity
    The epitope recognized by this monoclonal antibody is between amino acids 105-236. The antibody is expected to detect full length FOXP3 as well as both cleaved forms.
  • Tested applications
    Suitable for: IHC-P, IHC-Fr, WB, ICC/IF, Flow Cytmore details
  • Species reactivity
    Reacts with: Human, Cynomolgus monkey, Rhesus monkey
  • Immunogen

    FOXP3 fusion protein.

  • Positive control
    • WB: Human thymus and tonsil lysates; HEK-293T overexpressing Human FOXP3 cell lysate. ICC/IF: Head and neck squamous cell carcinoma cells IHC-P: Human tonsil, colon and breast cancer tissue; monkey spleen tissue.
  • General notes

    Abcam recommended secondaries - Goat Anti-Mouse HRP (ab205719) and Goat Anti-Mouse Alexa Fluor® 488 (ab150113).

    See other anti-mouse secondary antibodies that can be used with this antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab20034 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IHC-P Use a concentration of 10 µg/ml. Perform heat mediated antigen retrieval via the microwave method before commencing with IHC staining protocol.
IHC-Fr Use a concentration of 10 µg/ml.
WB Use a concentration of 4 - 5 µg/ml. Detects a band of approximately 50 kDa (predicted molecular weight: 47 kDa).
ICC/IF Use at an assay dependent concentration.
Flow Cyt Use 2µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

Target

  • Function
    Probable transcription factor. Plays a critical role in the control of immune response.
  • Involvement in disease
    Defects in FOXP3 are the cause of immunodeficiency polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) [MIM:304790]; also known as X-linked autoimmunity-immunodeficiency syndrome. IPEX is characterized by neonatal onset insulin-dependent diabetes mellitus, infections, secretory diarrhea, trombocytopenia, anemia and eczema. It is usually lethal in infancy.
  • Sequence similarities
    Contains 1 C2H2-type zinc finger.
    Contains 1 fork-head DNA-binding domain.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • AIID antibody
    • DIETER antibody
    • Forkhead box P3 antibody
    • Forkhead box protein P3 antibody
    • FOXP3 antibody
    • FOXP3_HUMAN antibody
    • FOXP3delta7 antibody
    • Immune dysregulation polyendocrinopathy enteropathy X linked antibody
    • Immunodeficiency polyendocrinopathy enteropathy X linked antibody
    • IPEX antibody
    • JM2 antibody
    • MGC141961 antibody
    • MGC141963 antibody
    • OTTHUMP00000025832 antibody
    • OTTHUMP00000025833 antibody
    • OTTHUMP00000226737 antibody
    • PIDX antibody
    • Scurfin antibody
    • XPID antibody
    see all

Images

  • FoxP3+ cells mainly accumulate centrally.

    The formalin-fixed, paraffin-embedded blocks were cut into approximately <2 µm thick slices and mounted on SuperFrost Plus microscope slides (Menzel Gläser, Braunschweig, Germany). After deparaffinization and rehydration, sections were immersed into Dako Target Retrieval solution (Dako North America Inc., Carpinteria, USA), pH 6, 1/10, incubated at 97°C–99°C at 750 Watt for 2x 15 minutes, and allowed to cool to room temperature for 20 minutes. Endogenous peroxidase activity was blocked by 10-minute incubation in 7.5% hydrogen-peroxide solution (Hydroxen Peroxide Solution, Sigma Aldrich Co., Munich, Germany). Immunohistochemical staining for FoxP3 (1/180 dilution; for 60 min) was performed according to standard procedure using MACH-3 mouse alkalic phosphatase polymer detection kit from Biocare Medical Systems (Concord, USA). The slides were incubated with monoclonal mouse antibody. Chromogen Red (Dako North Amerika Inc., Carpinteria, USA) was used as chromogen for FoxP3 staining, and lastly hematoxylin counterstaining was done (Vector Laboratories, Burlingam, USA).

  • Immunohistochemical analysis of human large and locally advanced breast cancers staining FOXP3 using ab20034. (a) Low level of FOXP3+, CTLA-4Treg infiltration (b) High level of FOXP3+ and CTLA-4Treg infiltration. (Itu: intratumoral Str: stromal)

  • All lanes : Anti-FOXP3 antibody [236A/E7] (ab20034) at 5 µg/ml

    Lane 1 : HEK293T cell lysate
    Lane 2 : HEk293T cell lysate overexpressing Human FOXP3
    Lane 3 : Human tonsil tissue lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 47 kDa



    ab20034 detects Human FOXP3 protein at ~50 kDa in HEK293T cells overexpressing the protein. It also detects FOXP3 in Human tonsil tissue lysate, however this band is significantly weaker in endogenous conditions. Upon higher exposure, weak bands can also be observed in HEK293T cell lysate.

    This blot was produced using a 4-12% Bis-Tris gel under the MOPS buffer system. The gel was run at 200V for 60 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with Anti-FOXP3 antibody [236A/E7] (ab20034; 5 microgram per mL) overnight at 4°C. Antibody binding was detected using infrared labelled goat anti-mouse (green; 1:10000) for 1 hour at room temperature before imaging.

  • ab20034 staining FOXP3 in human colon tissue section by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue underwent formaldehyde fixation before heat mediated antigen retrieval and then blocking with 10% serum for 2 hours at 21°C. The primary antibody was diluted 1/50 and incubated with sample for 2 hours at 21°C. An Alexa Fluor®488-conjugated rabbit polyclonal to mouse IgG was used undiluted as secondary antibody.

    See Abreview

  • ab20034 staining FOXP3 in Cynomolgus Monkey Spleen tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 10% donkey serum for 20 minutes at room temperature; antigen retrieval was by heat mediation using EDTA, pH9.0. Samples were incubated with primary antibody (1/100) for 30 minutes. A Biotin-conjugated Donkey anti-mouse polyclonal (1/2000) was used as the secondary antibody.

    See Abreview

  • ab20034 used on paraffin embedded human tonsil sections.
    Double staining: CD30 in red, FOXP3 in black, blue is haemotoxylin counterstaining of nuclei.

    As expected FOXP3 stains a sub-population of T cells.

  • Anti-FOXP3 antibody [236A/E7] (ab20034) at 5 µg/ml + Human thymus at 20 µg

    Secondary
    IR-labelled goat anti-mouse (green; 800) at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size: 47 kDa
    Observed band size: 50 kDa
    why is the actual band size different from the predicted?



    This blot was produced using a 4-12% Bis-Tris gel under the MOPS buffer system. The gel was run at 200V for 60 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with Anti-FOXP3 antibody [236A/E7] (ab20034; 5 microgram per mL), overnight at 4°C. Antibody binding was detected using infrared labelled goat anti-mouse (green; 1:10000) for 1 hour at room temperature before imaging.

  • Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized head and neck squamous cell carcinoma cells stained for FOXP3 (green) using ab20034 at 1/100 dilution in ICC/IF.

    See Abreview

  • ab20034 used on paraffin embedded human tonsil sections.
    FOXP3 staining is black, blue is haemotoxylin counterstaining of nuclei.

    As expected FOXP3 stains a sub-population of T cells.

  • IHC image of Foxp3 staining in Human normal tonsil formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab20034, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

    *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

  • ab20034 staining FOXP3 in Human breast cancer tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with acetone/methanol (1:2), permeabilized with TBS-TritonX-100 and blocked with 10% serum for 10 minutes at 20°C. Samples were incubated with primary antibody (1/50 in PBT (PBS/BSA/Tween)) for 30 minutes at 20°C. An undiluted HRP-conjugated rat anti-mouse/rabbit IgG polyclonal was used as the secondary antibody.

    See Abreview

References

This product has been referenced in:
  • Kaewkangsadan V  et al. Tumour-draining axillary lymph nodes in patients with large and locally advanced breast cancers undergoing neoadjuvant chemotherapy (NAC): the crucial contribution of immune cells (effector, regulatory) and cytokines (Th1, Th2) to immune-mediated tumour cell death induced by NAC. BMC Cancer 18:123 (2018). IHC-P ; Human . Read more (PubMed: 29390966) »
  • Heby M  et al. Relationship between mismatch repair immunophenotype and long-term survival in patients with resected periampullary adenocarcinoma. J Transl Med 16:66 (2018). IHC ; Human . Read more (PubMed: 29540182) »
See all 214 Publications for this product

Customer reviews and Q&As

1-10 of 32 Abreviews or Q&A

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (Brain)
Permeabilization
No
Specification
Brain
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 21°C
Fixative
Formaldehyde

Ms. Ji Hye Han

Verified customer

Submitted Oct 22 2018

Application
Western blot
Sample
Mouse Tissue lysate - whole (Brain)
Gel Running Conditions
Non-reduced Non-Denaturing (Native) (10%)
Loading amount
30 µg
Treatment
250 μg/kg LPS for 1 week
Specification
Brain
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Ms. Ji Hye Han

Verified customer

Submitted Oct 22 2018

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Tonsil)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Ventana CC1 (EDTA pH8.5)
Permeabilization
No
Specification
Tonsil
Blocking step
Ventana blocking agent as blocking agent for 4 minute(s) · Concentration: 100% · Temperature: 36°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Sep 25 2018

Application
Immunohistochemistry (Frozen sections)
Sample
Human Tissue sections (Human Skin)
Permeabilization
Yes - 0.5% Triton TBS Ca/Azide
Specification
Human Skin
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 37°C
Fixative
Paraformaldehyde

Mr. Tyler Crowe

Verified customer

Submitted Aug 21 2018

Application
Immunohistochemistry (Frozen sections)
Sample
Human Tissue sections (Lymph node)
Permeabilization
No
Specification
Lymph node
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 21°C
Fixative
Acetone

Dr. Christian Ostalecki

Verified customer

Submitted May 24 2018

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (liver)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: dako retrieval buffer
Permeabilization
No
Specification
liver
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Jan 02 2018

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (B16 tumors taken out of C57bl mice)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate pH 6
Permeabilization
No
Specification
B16 tumors taken out of C57bl mice
Blocking step
Innovex Background Buster as blocking agent for 30 minute(s) · Concentration: 100% · Temperature: 4°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Sep 18 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (EBV-associated gastric cancer)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: CC1(Ventana EDTA)
Permeabilization
No
Specification
EBV-associated gastric cancer
Fixative
10% neutral formalin

Abcam user community

Verified customer

Submitted Apr 27 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Tonsil)
Permeabilization
No
Specification
Tonsil
Blocking step
H2O2 as blocking agent for 10 minute(s) · Concentration: 3%
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Dec 12 2016

Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Mouse Tissue sections (Spleen)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: 10 mM sodium citrate
Specification
Spleen
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Jun 03 2016

1-10 of 32 Abreviews or Q&A

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