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PhosphoSitePlus® Resource

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REACTIVITY SENSITIVITY MW (kDa) Isotype
M Endogenous 45 Rabbit IgG
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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded 4T1 metastatic tumor in mouse lung using FoxP3 (D6O8R) Rabbit mAb performed on the Leica® Bond™ Rx.

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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded mouse thymus using FoxP3 (D6O8R) Rabbit mAb.

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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded mouse spleen using FoxP3 (D6O8R) Rabbit mAb.

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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded mouse small intestine using FoxP3 (D6O8R) Rabbit mAb.

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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded mouse lung using FoxP3 (D6O8R) Rabbit mAb.

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Immunofluorescence (Frozen)

Immunofluorescent analysis of mouse spleen using FoxP3 (D6O8R) Rabbit mAb (red) and a CD4 antibody (green). Blue = DRAQ5® #4084 (fluorescent DNA dye).

Note: FoxP3 (D6O8R) Rabbit mAb has been validated on freshly-prepared frozen tissue sections that were fixed for 15 minutes in 4% formaldehyde. Over-fixation diminishes the performance of this antibody.

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Flow Cytometry

Flow cytometric analysis of murine spleen lymphocytes using a CD25 antibody and FoxP3 (D6O8R) Rabbit mAb. Anti-rabbit IgG (H+L), (F(ab')2 ) Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody. Analysis was performed on a gated CD4+ T cell population.

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Immunohistochemistry (Leica® Bond)

NOTE: Please see product datasheet or product webpage for appropriate antibody dilution.

  Step Reagents Time/Temperature

1.

Dewax

Bond Dewax Solution, 100% Alcohol, Bond Wash Solution

Pre-programmed Leica®Bond

2.

Antigen Retrieval

Bond Epitope Retrieval ER2 Solution

20 min., 100°C

3.

Peroxide Blocking

Peroxide Block*

5 min.

 

WASH

Bond Wash Solution

3x 0:00 min.

4.

Protein Block

CST #5425 NGS or #15019 Animal-Free Blocking Solution

20 min.

5.

Primary Antibody

Dilute in #8112 Antibody Diluent

30 min.

 

WASH

Bond Wash Solution

3x 2:00 min.

6.

Secondary Detection

Novolink Polymer*

10 min.

 

WASH

Bond Wash Solution/Deionized Water

Custom (see below)

7a.

Visualization

Mixed DAB Refine*

0:00 min.

7b.

Visualization

Mixed DAB Refine*

10 min.

 

WASH

Deionized Water

3x 0:00 min.

8.

Counterstain

Hematoxylin*

5 min.

 

WASH

Deionized Water

3x 0:00 min.

 

WASH

Bond Wash Solution

3x 0:00 min.

 

WASH

Deionized Water

3x 0:00 min.

9.

Dehydration (Offline):
Incubate sections in 95% ethanol two times for 10 sec. each.
Repeat in 100% ethanol, incubating sections two times for 10 sec each.
Repeat in xylene, incubating sections two times for 10 sec each.
Mount sections with coverslips and mounting medium (#14177).

  Custom wash: Bond Wash Solution 2:00
    Bond Wash Solution Dispenser Type: OPEN 0:00
    Bond Wash Solution 2:00
    Bond Wash Solution Dispenser Type: OPEN 0:00
    Bond Wash Solution 0:00
    Deionized Water 0:00

*Reagent included in Bond Polymer Refine Detection Kit
Catalog No: DS9800

LEICA® is a registered trademark of Leica®Microsystems IR GmbH.

Bond is a trademark of Leica®Biosystems Melbourne Pty. Ltd.

No affiliation or sponsorship between CST and Leica®Microsystems IR GmbH or Leica®Biosystems Melbourne Pty. Ltd. is implied.

posted February 2017

Protocol Id: 1444

Immunohistochemistry (Paraffin)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. Xylene.
  2. Ethanol, anhydrous denatured, histological grade (100% and 95%).
  3. Deionized water (dH2O).
  4. Hematoxylin (optional).
  5. Wash Buffer:
    1. 1X Tris Buffered Saline with Tween® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween® 20 (#9997) to 900 ml dH20, mix.
  6. SignalStain® Antibody Diluent (#8112).
  7. 1X Citrate Unmasking Solution: To prepare 250 mL of 1X citrate unmasking solution, dilute 25 ml of SignalStain® Citrate Unmasking Solution (10X) (#14746) with 225 mL of dH2O.
  8. 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H2O2 to 90 ml dH2O.
  9. Blocking Solution: TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
    1. TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425).
    2. 1X Animal-Free Blocking Solution: to 4 mL of dH2O add 1 ml of Animal-Free Blocking Solution (5X) (#15019).
  10. Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Rabbit #8114).
  11. Substrate: SignalStain® DAB Substrate Kit (#8059).
  12. Hematoxylin: Hematoxylin (#14166).
  13. Mounting Medium: SignalStain® Mounting Medium (#14177).

B. Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

  1. Deparaffinize/hydrate sections:
    1. Incubate sections in three washes of xylene for 5 min each.
    2. Incubate sections in two washes of 100% ethanol for 10 min each.
    3. Incubate sections in two washes of 95% ethanol for 10 min each.
  2. Wash sections two times in dH2O for 5 min each.

C. Antigen Unmasking

For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

D. Staining

  1. Wash sections in dH2O three times for 5 min each.
  2. Incubate sections in 3% hydrogen peroxide for 10 min.
  3. Wash sections in dH2O two times for 5 min each.
  4. Wash sections in wash buffer for 5 min.
  5. Block each section with 100–400 µl of preferred blocking solution for 1 hr at room temperature.
  6. Remove blocking solution and add 100–400 µl primary antibody diluted in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
  7. Equilibrate SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) to room temperature.
  8. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
  9. Cover section with 1–3 drops SignalStain® Boost Detection Reagent (HRP, Rabbit #8114) as needed. Incubate in a humidified chamber for 30 min at room temperature.
  10. Wash sections three times with wash buffer for 5 min each.
  11. Add 1 drop (30 µl) SignalStain® DAB Chromogen Concentrate to 1 ml SignalStain® DAB Diluent and mix well before use.
  12. Apply 100–400 µl SignalStain® DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
  13. Immerse slides in dH2O.
  14. If desired, counterstain sections with hematoxylin (#14166).
  15. Wash sections in dH2O two times for 5 min each.
  16. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 sec each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 sec each.
    3. Repeat in xylene, incubating sections two times for 10 sec each.
  17. Mount sections with coverslips and mounting medium (#14177).

posted February 2010

revised March 2016

Protocol Id: 283
Page

Immunofluorescence (Frozen)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
  3. Blocking Buffer: (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425) to 9.5 ml 1X PBS) and mix well. While stirring, add 30 µl Triton™ X-100.
  4. Antibody Dilution Buffer: (1X PBS / 1% BSA / 0.3% Triton™ X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (#9998), mix.
  5. Recommended Fluorochrome-conjugated Anti-Rabbit secondary antibodies:

    NOTE: When using any primary or fluorochrome-conjugated secondary antibody for the first time, titrate the antibody to determine which dilution allows for the strongest specific signal with the least background for your sample.

  6. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Frozen/Cryostat Sections (IF-F)

  1. For fixed frozen tissue proceed with Immunostaining (Section C).
  2. For fresh, unfixed frozen tissue, please fix immediately, as follows:
    1. Cover sections with 4% formaldehyde dilute in 1X PBS.

      NOTE: Formaldehyde is toxic, use only in fume hood.

    2. Allow sections to fix for 15 minutes at room temperature.
    3. Aspirate liquid, rinse three times in 1X PBS for 5 minutes each.
    4. Proceed with Immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

  1. Block specimen in Blocking Buffer for 60 min.
  2. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 min each.
  6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hr at room temperature in the dark.
  7. Rinse three times in 1X PBS for 5 min each.
  8. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

posted November 2006

revised July 2016

Protocol Id: 151
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Flow Cytometry

A. Solutions and Reagents

NOTE: Prepare solutions with RODI (reverse osmosis deionized) or equivalent grade water.

B. Fixation

NOTE: Surface staining of CD4 and CD25 antibodies should be performed on live cells prior to fixation/permeabilization, as per manufacturer’s requirements.

  1. Disaggregate spleens using a 100 µm nylon mesh cell strainer and collect in cold PBS.
  2. Wash cells by centrifugation and resuspend in 5 ml ACK Lysis Buffer for 5 mins. (Red Blood Cell lysis).
  3. Wash by centrifugation in Incubation Buffer.
  4. Resuspend freshly isolated murine spleen cells at 5–10 x 105 cells in 100 µl Incubation Buffer per assay tube.
  5. Add manufacturer's recommended volume of CD25 and CD4 surface marker antibodies to live cells in each sample tube.mix and let incubate for at least 30 min, on ice.
  6. Wash cells by centrifugation and aspirate supernatant.
  7. Resuspend cells in 500 µl of 2% formaldehyde.
  8. Fix for 15 min at room temperature.
  9. Wash 2X by centrifugation in Incubation Buffer.

C. Permeabilization

  1. Add 1ml of 0.3% Triton™ X-100 (v/v in PBS) to the cell pellet.
  2. Vortex and let stand for 30 min at room temperature.
  3. Wash 2X by centrifugation in Incubation Buffer.

D. Immunostaining

  1. Resuspend cell pellets in 100 µl of FoxP3 (D6O8R) XP® Rabbit mAb #12653 working solution (Stock antibody diluted 1:200 in Incubation Buffer).
  2. Incubate for 1 hr at room temperature.
  3. Wash 2X by centrifugation in Incubation Buffer.
  4. Resuspend cells in fluorochrome-conjugated secondary antibody diluted in Incubation Buffer at the recommended dilution.
  5. Incubate for 30 min at room temperature.
  6. Wash 2X by centrifugation in Incubation Buffer.
  7. Resuspend cells in 350 µl of Incubation Buffer and analyze on flow cytometer.
  8. FoxP3 can be plotted against CD25 on a bivariate scattergram gated on CD4+ T lymphocytes.

posted June 2013

Protocol Id: 62

Product Usage Information

Application Dilutions
IHC-Leica® Bond™ 1:400
Immunohistochemistry (Paraffin) 1:100
Immunofluorescence (Frozen) 1:100
Flow Cytometry 1:50

Storage: Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

FoxP3 (D6O8R) Rabbit mAb recognizes endogenous levels of total FoxP3 protein. This antibody recognizes mouse FoxP3 protein and is also reactive with human FoxP3; however, this antibody is not suggested for immunohistochemical analysis of human tissues. Instead, FoxP3 (D2W8E™) Rabbit mAb (IHC Specific) #98377 is recommended for IHC analysis of human tissue samples.


Species Reactivity: Mouse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro44 of mouse FoxP3 protein.

Forkhead box (Fox) proteins are a family of evolutionarily conserved transcription factors containing a sequence known as Forkhead box or winged helix DNA binding domain (1). The human genome contains 43 Fox proteins that are divided into subfamilies. The FoxP subfamily has four members, FoxP1 - FoxP4, which are broadly expressed and play important roles in organ development, immune response and cancer pathogenesis (2-4). The FoxP subfamily has several characteristics that are atypical among Fox proteins: their Forkhead domain is located at the carboxy-terminal region and they contain motifs that promote homo- and heterodimerization. FoxP proteins usually function as transcriptional repressors (4,5).

FoxP3 is crucial for the development of T cells with regulatory properties (Treg) (6). Mutations in FoxP3 are associated with immune dysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome (IPEX) (7), while overexpression in mice causes severe immunodeficiency (8). Research studies have shown that FoxP3 functions as a tumor suppressor in several types of cancer (9-11).


1.  Myatt, S.S. and Lam, E.W. (2007) Nat Rev Cancer 7, 847-59.

2.  Shu, W. et al. (2001) J Biol Chem 276, 27488-97.

3.  Lu, M.M. et al. (2002) Gene Expr Patterns 2, 223-8.

4.  Koon, H.B. et al. (2007) Expert Opin Ther Targets 11, 955-65.

5.  Li, S. et al. (2004) Mol Cell Biol 24, 809-22.

6.  Ochs, H.D. et al. (2007) Immunol Res 38, 112-21.

7.  Bennett, C.L. et al. (2001) Nat Genet 27, 20-1.

8.  Kasprowicz, D.J. et al. (2003) J Immunol 171, 1216-23.

9.  Zuo, T. et al. (2007) Cell 129, 1275-86.

10.  Zuo, T. et al. (2007) J Clin Invest 117, 3765-73.

11.  Wang, L. et al. (2009) Cancer Cell 16, 336-46.


Entrez-Gene Id 50943
Swiss-Prot Acc. Q9BZS1


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
BOND is a trademark of Leica Biosystems Melbourne Pty. Ltd. No affiliation or sponsorship between CST and Leica Microsystems IR GmbH or Leica Biosystems Melbourne Pty. Ltd is implied.
LEICA is a registered trade​mark of Leica Microsystems IR GmbH.

12653
FoxP3 (D6O8R) Rabbit mAb