Forkhead box protein P3 Antibodies

Forkhead box protein P3 (Foxp3)

Transcriptional regulator which is crucial for the development and inhibitory role of regulatory T-cells (Treg). Plays an important role in maintaining homeostasis of the immune system by permitting the acquisition of full suppressive function and stability of the Treg lineage, and by directly regulating the expansion and role of conventional T-cells.

Can act either as a transcriptional repressor or a transcriptional activator depending on its interactions with other transcription factors, histone acetylases and deacetylases. The suppressive activity of Treg involves the coordinate activation of many genes, such as CTLA4 and TNFRSF18 by FOXP3 along with repression of genes encoding cytokines such as interleukin-2 (IL2) and interferon- gamma (IFNG).

Gene Information

Mouse
Gene Name:	Foxp3
Uniprot:	Q99JB6
Entrez:	20371

Family

Belongs to:
No superfamily

Alternative Names

AIID; AIIDMGC141961; human foxp3; mouse foxp3; DIETER; forkhead box P3; Forkhead Box Protein P3; foxp3 ihc; foxp3 paraffin; FoxP3; FOXP3delta7; immune dysregulation, polyendocrinopathy, enteropathy, X-linked; Immunodeficiency, Polyendocrinopathy, Enteropathy, X-Linked; IPEX; JM2; MGC141961; MGC141963; PIDX; PIDXMGC141963; SCURFIN; XPID; XPIDpolyendocrinopathy, enteropathy, X-linked

Molecular Weight

Mass (kDA):
47.346 kDA

Genomic Context

Mouse
Location:	X A1.1|X 3.41 cM
Sequence:	X;

Tissue Specificity

High level of expression in thymus and spleen.

Diseases

• Autoimmune Reaction
• Inflammation
• Neoplasms
• Autoimmune Diseases
• Malignant Neoplasms
• Autoimmunity
• Decreased Immunologic Activity [pe]
• Diabetes Mellitus
• Carcinoma
• Diabetes Mellitus, Insulin-dependent
• Immunologic Tolerance
• Graft-vs-host Disease
• Intestinal Diseases

• Experimental Autoimmune Encephalomyelitis
• Colitis
• Arthritis
• Asthma
• Infective Disorder
• Encephalomyelitis
• Allergy

Pathways

• Immune Response
• Pathogenesis
• Secretion
• Cell Proliferation
• Cytokine Production
• T Cell Proliferation
• Cell Activation
• Cell Differentiation
• T Cell Activation
• Cell Development
• Tolerance Induction
• Hypersensitivity
• Inflammatory Response

• Sensitization
• T Cell Differentiation
• Cytokine Secretion
• Cell Death
• Localization
• Methylation
• Lymphocyte Proliferation

PTMs

• Phosphorylation
• Methylation
• Demethylation
• Acetylation
• Cleavage
• Deamidation
• Deacetylation
• Ribosylation
• Reduction

• Dephosphorylation
• Ubiquitination
• Decarboxylation
• Farnesylation
• Glycosylation
• Geranylgeranylation
• Sumoylation
• Citrullination
• Cysteinylation

• Biotinylation

Research Areas

• Adaptive Immunity
• Cell Biology
• Chromatin Research
• Immunology
• Regulatory Immunology

• Transcription Factors and Regulators

For details, visit:
bosterbio.com/bosterbio-gene-info-cards/Foxp3

Best Uses Of The FOXP3 Marker

A monoclonal antibody to Foxp3 can also be used in immunofluorescence as well as immunohistochemistry studies (IHC-P). The monoclonal anti CD31/PECAM-1 antibody reacts with human, rabbit and monkey tissues. It is also available in Endothobody form, which is a validator of antibodies against positive or negative samples.

Staining that is optimal for Foxp3

In the current study, we have discovered an intracellular staining technique to detect Foxp3 within human cells. This protocol employs paraformaldehyde a fixative and saponin as a membrane permeating reagent. This method can be utilized to detect nuclear FP molecules and those in the cytosol at the same time. Particularly, we found that 259D/C7 staining performed better on fresh cells compared to frozen cells.

Mouse regulatory T cells express FoxP3, a transcriptional factor that regulates Treg function. Because FoxP3 is found intracellularly it is not able to stain the nucleus of the cell with the majority of antibodies. Contrarily the FoxP3+ Treg clone that was created by eBioscience demonstrates bright staining when applied to cells. This technique is designed to detect functional Tregs.

Flow cytometry-based immunostaining of human Tregs is a superior method to identify these cells. The current study has refined the FOXP3 staining procedure to be compatible with both antigen-specific as well as polyclonal human Tregs. The three methods all produced similar amounts of FOXP3+ cells counts. The eBioscience and Fluidigm protocols resulted in similar proportions of FOXP3+ cells which indicates that they are equally sensitive and specific to detect human Tregs.

Optimal staining for Foxp3, also known as FITC, is done using intracellular fixative/permeabilization reagents. These reagents permit intracellular detection and permeabilization of the Foxp3 nuclear proteins in T regulatory cells. Not surprisingly, the YFP signal disappears following Fix/Perm treatment. This could be a result of leakiness in the cell membrane.

Foxp3 detection is accomplished using a primary antibody that is designated MF-14. The primary antibody should also be dilute with "antibody buffer" (adb) to improve staining. After the primary antibodyhas been added, you should add Mowiol and mounting medium to the slides. If you are using GFP or FITC Do not use the primary antibody unless you are sure of the species.

Validated antibodies that have been raised in various species can decrease cross-reactivity in signaling processing. This advantage isn't available for all primary antibodies. For instance when you use GITR primary antibody to detect Foxp3, only 25% of cells were colocalized with the protein. If you're testing for Foxp3 in human cells, be sure to test the primary antibody against the corresponding protein using GITR as a housekeeping gene.

Staining optimally to PECAM-1

To maximize the capacity of PET-CT to detect the presence of PECAM-1 on human tissues, the company recommends the use of a two-step visualization system called Ultraview. However, this method failed to provide the most effective staining. FOXP3-specific antibodies showed positive staining reactions in all cases. FOXP3 has been linked to specific types of tissue, such as bone or cartilage.

Choose a mmAb that is suitable for the CD31-PECAM-1 antibody immunostaining. The best JC70A antibody can detect CD31 in alkaline buffer as well as in multimer-based detection techniques that use three steps. It should not be used to detect sinusoidal endothelial cell. Furthermore, this method shows moderate to weak staining in endothelial cells lining large blood vessels.

The CD31/PECAM-1 antibody recognizes human CD31 at a variety of molecular sizes. It is mapped to a region comprising 700- 738 human CD31. Additionally it also binds recombinant human or porcine CD31 and is detected in flow cytometry. The concentration of this antibody should be adjusted as per your needs. A citrate buffer can be used when you have images of high resolution of a specific cell type to enhance staining.

It is possible to employ an array of markers to detect Tregs using the FOXP3 marker. These markers can be used in either live or fixed cells. There is no requirement to permeabilize cells for staining, making them appropriate for downstream applications. This method is particularly useful to identify and stain Tregs. This staining technique works on both live and fixed cells and does not require cell permeabilization.

Another monoclonal antibody reacts to mouse CD31 which is also known as PECAM-1. This molecule is expressed on the surface of the majority of endothelial cells and is a key component in intercellular junctions. PECAM-1 is involved in transendothelial migration. It is also expressed by bone marrow-derived embryonic stem cells and stem cells derived from hematopoietic hematopoietic.

CD31 is a transmembrane glycoprotein high-yielding and is a sign of endothelial cell function. Antibodies that recognize CD31 react specifically with murine cells and exhibit no cross-reactivity with human CD31. In the end, CD31 antibodies are the gold standard in studies using mouse models. The antibody has been validated by a pathologist to give an accurate diagnosis.

Staining optimally for CD31

To ensure optimal staining of CD31 with the FOXP3 marker we employed the plasmid created by our laboratory. The plasmid carries four sets of primers that flank the putative FOXP3 binding sites located in the promoter region of VEGF. The optimal titration of antibodies and fix/perm buffers must be used to ensure the best staining of CD31 with the FOXP3 marker. We also used an anti-FOXP3 primary antibody in combination with a control human IgG to detect VEGF. The analysis of DNA fragments was then normalized against genomic DNA.

The FOXP3 marker, also known as PECAM-1, is found on T cells that are a subset. It is believed to be present on recent emigrating cells but is lost during the post-thymic expansion of the peripheral periphery. In our experiments, CD31+FoxP3+CD4+ T cells were limited to the CD45RA+FoxP3lo group that indicates recent thymic migration.

The SZ31 Clone is an immunohistochemical reagent that binds specifically to murine CD31. It is perfect for research using mouse models since it exhibits almost no cross-reactivity to human CD31. It is considered to be a high-quality standard for endothelial staining in mouse paraffin sections. With just one antibody, you are able to conduct an in-depth study of tumor angiogenesis with high levels of confidence.

Another example of vascular soft tissue sarcomas is angiosarcoma. Both vascular and nonvascular tumors show positive staining of CD31. In epithelioid hemangioendothelioma, CD31 and ERG were positively stained. The results from this experiment suggest the presence of both vascular tumors.

FOXP3 is also involved in the regulation of angiogenesis in breast cancer. The inhibition of breast cancer angiogenesis via FOXP3 is also associated with suppressing breast cancer angiogenesis. To better understand tumor angiogenesis, it is essential to stain CD31 with the FOXP3 marker.

Tissue incubation is necessary to ensure the best staining of CD31 with the FOXP3 marker. The first step is to identify the primary antibody by an Qdot800-labeled second antibody. If the primary antibody is not labeled the indirect detection method is an alternative. The tissues were first incubated at room temperatures, then incubated with remaining antibodies for the remainder of the night at 4degC.