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Fluorescent Multiplex Immunohistochemistry

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

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using FoxP3 (D2W8E™) Rabbit mAb (IHC Specific).

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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded COS-7 cell pellets, untransfected (left) or transfected with human FoxP3 protein (right), using FoxP3 (D2W8E™) Rabbit mAb (IHC Specific).

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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded human tonsil using FoxP3 (D2W8E™) Rabbit mAb (IHC Specific).

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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

Product Usage Information

Application Dilutions
Immunohistochemistry (Paraffin) 1:200

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 (D2W8E™) Rabbit mAb (IHC Specific) recognizes endogenous levels of total FoxP3 protein.


Species Reactivity: Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant protein specific to human FoxP3.

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.
SignalStain is a trademark of Cell Signaling Technology, Inc.

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FoxP3 (D2W8E™) Rabbit mAb (IHC Specific)