FOXD3 Antibody

What is FOXD3 and what are its primary functions in cellular biology?

FOXD3 is a transcription factor belonging to the forkhead gene family. It primarily functions as a transcriptional regulator that binds to the consensus sequence 5'-A[AT]T[AG]TTTGTTT-3' and can act as both a transcriptional repressor and activator depending on cellular context .

FOXD3 serves several critical biological functions:

• Maintenance of pluripotency in pre-implantation and peri-implantation embryonic stages

• Promotion of neural crest cell development from neural tube progenitors

• Restriction of neural progenitor cells to the neural crest lineage while suppressing interneuron differentiation

• Mediation of fate restriction choices for multipotent neural crest progenitors

• Heterochromatin-mediated repression of repeat elements in mouse embryonic stem cells

In stem cells, FOXD3 exhibits a bimodal role, functioning either as an activator or repressor through mechanisms such as enhancer decommissioning and recruitment of chromatin modulators including histone demethylase LSD1, chromatin remodeling factor BRG1, and histone deacetylases (HDACs) .

What is the molecular structure and cellular localization of FOXD3?

FOXD3 is a protein with the following characteristics:

The protein contains a DNA-binding forkhead domain that enables its function as a transcription factor, and its N-terminal region (aa 1-140) is commonly used as an immunogen for antibody production .

What criteria should be considered when selecting a FOXD3 antibody for specific research applications?

When selecting a FOXD3 antibody, researchers should consider several critical parameters:

Species Reactivity:
Different antibodies show varying reactivity profiles. Commercial antibodies are available with reactivity to human, mouse, rat, chicken, monkey, and other species. Ensure the antibody reacts with your species of interest .

Application Compatibility:
Verify antibody validation for your specific application:

• Western Blot (WB): Most FOXD3 antibodies are validated for WB with recommended dilutions ranging from 1:500-1:2000

• Immunohistochemistry (IHC): Dilutions typically range from 1:10-1:1000, with paraffin-embedded sections requiring specific protocols

• ELISA: Often using higher dilutions (1:10000)

• Chromatin Immunoprecipitation (ChIP): Specialized antibodies validated for chromatin binding studies

• Immunofluorescence (IF): Particularly important for co-localization studies

Antibody Format:

• Monoclonal vs. Polyclonal: Monoclonal antibodies (like clone 5G9) offer higher specificity, while polyclonal antibodies may provide stronger signals

• Host Species: Consider potential cross-reactivity issues, especially when working with mouse samples using mouse-derived antibodies

• Conjugates: Unconjugated vs. fluorescent or enzyme-conjugated antibodies based on detection method

Validated Performance:
Prioritize antibodies with published validation data, including Western blots showing the expected band size (approximately 50-60 kDa for FOXD3) and immunohistochemistry images demonstrating appropriate cellular localization .

How can researchers validate FOXD3 antibody specificity for their experimental systems?

Proper validation of FOXD3 antibodies is essential to ensure experimental rigor. Consider the following comprehensive validation strategy:

Positive and Negative Controls:

• Positive controls: Cell lines with known FOXD3 expression such as NTERA-2, Jurkat, and embryonic stem cells

• Negative controls: Cell lines with limited FOXD3 expression (multiple non-neural crest derived cell lines)

• Genetic controls: Compare wild-type samples with FOXD3 knockout or knockdown samples

Multiple Detection Methods:

• Western blot validation should show a single specific band at ~47-60 kDa

• Validation across multiple applications (WB, IHC, IF) ensures consistent results

• Peptide competition assays can confirm binding specificity

Application-Specific Controls:

• For ChIP experiments: Include IgG controls and validate with known FOXD3 binding sites

• For IHC/IF: Use blocking peptides or secondary antibody-only controls

• For mouse tissues using mouse antibodies: Use mouse-on-mouse blocking reagents to reduce background

Published data indicates that when working with mouse samples, mouse blocking reagent may be needed for IHC and ICC experiments to reduce high background signal. These reagents can be found under catalog numbers PK-2200-NB and MP-2400-NB in some commercial sources .

What are the optimal protocols for using FOXD3 antibodies in Western blot analyses?

Sample Preparation:

• Lyse cells in an appropriate buffer (e.g., RIPA buffer with protease inhibitors)

• For nuclear proteins like FOXD3, nuclear extraction protocols may yield better results

• Quantify protein concentration using Bradford or BCA assay

• Recommended loading amount: 20-40 μg of total protein extract

Electrophoresis and Transfer:

• Separate proteins on 10-12% SDS-PAGE gels

• Transfer to PVDF membrane (preferred over nitrocellulose for FOXD3)

• Confirm transfer efficiency with reversible staining (Ponceau S)

Antibody Incubation and Detection:

• Block membrane in 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Incubate with primary FOXD3 antibody at optimal dilution (typically 1:500-1:2000) overnight at 4°C

• Wash 3× with TBST, 5 minutes each

• Incubate with appropriate HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour at room temperature

• Wash 3× with TBST, 5 minutes each

• Visualize using ECL detection system

Validation Controls:
Include positive controls such as FOXD3-transfected HEK293 cells , and negative controls such as mock-transfected cells. Expected band size is approximately 47-60 kDa depending on post-translational modifications .

Troubleshooting Tips:

• If detecting multiple bands, increase antibody dilution or optimize blocking conditions

• For weak signals, longer exposure times or signal enhancement reagents may be required

• Nuclear extraction may improve detection if whole cell lysates show weak signals

How should FOXD3 antibodies be optimized for immunohistochemistry and immunofluorescence applications?

Tissue Preparation:

• Fix tissues in 4% paraformaldehyde or 10% neutral buffered formalin

• Process and embed in paraffin or prepare frozen sections (8-10 μm thickness)

• For paraffin sections: Deparaffinize and perform antigen retrieval (citrate buffer pH 6.0 or EDTA buffer pH 9.0, depending on antibody recommendations)

Immunohistochemistry Protocol:

• Block endogenous peroxidase activity with 3% H₂O₂ (if using HRP detection)

• Block non-specific binding with 5-10% normal serum from secondary antibody species

• Incubate with primary FOXD3 antibody at optimized dilution (typical range: 1:200-1:1000 for paraffin sections)

• Wash 3× with PBS

• Incubate with appropriate biotinylated secondary antibody

• Apply avidin-biotin complex (ABC) and develop with DAB substrate

• Counterstain, dehydrate, and mount

Immunofluorescence Protocol:

• Block non-specific binding with 5-10% normal serum

• Incubate with primary FOXD3 antibody (typical dilution 1:100-1:500)

• Wash 3× with PBS

• Incubate with fluorophore-conjugated secondary antibody

• Counterstain nuclei with DAPI (1:5000)

• Mount with anti-fade mounting medium

Critical Optimization Parameters:

• Antibody dilution: Begin with manufacturer's recommendation and optimize as needed

• Antigen retrieval method: Compare citrate and EDTA-based methods

• Incubation time and temperature: Overnight at 4°C vs. 1-3 hours at room temperature

• Signal amplification: Consider tyramide signal amplification for weak signals

Special Considerations:
For sequential detection of FOXD3 and other markers (such as p75), published protocols recommend incubating sections with anti-FOXD3 and Cy3-conjugated secondary antibody first, followed by unconjugated anti-rabbit IgG (1:15) before immunodetection of the second marker .

What are the best practices for using FOXD3 antibodies in chromatin immunoprecipitation (ChIP) assays?

ChIP assays are crucial for determining FOXD3 binding sites on chromatin. The following protocol has been successfully used in published FOXD3 research:

Chromatin Preparation:

• Cross-link cells with 1% formaldehyde for 10 minutes at room temperature

• Quench with 0.125 M glycine for 5 minutes

• Lyse cells and isolate nuclei

• Sonicate chromatin to fragments of 200-500 bp

• Pre-clear chromatin with protein A/G beads

Immunoprecipitation:

• Incubate chromatin with 2-5 μg of validated FOXD3 antibody overnight at 4°C

• Include negative control (IgG from same species as FOXD3 antibody)

• Add protein A/G beads and incubate for 2-3 hours

• Wash beads with increasingly stringent buffers

• Elute complexes and reverse cross-links

DNA Analysis:

• Purify DNA and analyze by qPCR for specific targets or sequence by NGS

• For FOXD3, known binding sites include MERVL-LTR, MERVL-int, and MSR regions

Validation Controls:

• Input control: 5-10% of starting chromatin

• IgG control: Non-specific antibody of same isotype

• Positive control loci: Known FOXD3 binding sites (consensus sequence 5'-A[AT]T[AG]TTTGTTT-3')

• Negative control loci: Regions not expected to bind FOXD3 (e.g., L1MdA)

Research has demonstrated that FOXD3 binds to and represses MERVL and major satellite repeats (MSRs) in mouse embryonic stem cells. ChIP-qPCR with FOXD3 antibodies shows significant enrichment over MERVL-LTR, MERVL-int, and MSR, but not over L1MdA regions .

How can FOXD3 antibodies be used to study neural crest stem cell development and differentiation?

FOXD3 antibodies are invaluable tools for investigating neural crest development due to FOXD3's critical role in maintaining multipotency of neural crest stem cells (NCSCs).

Experimental Approaches:

• Developmental Expression Profiling:

  • Use FOXD3 antibodies in combination with NCSC markers (Sox10, p75) for co-immunostaining to track developmental stages

  • FOXD3 expression is maintained in migratory cranial NC at 8.5 dpc and becomes restricted to presumptive cranial and dorsal root ganglia by 9.5 dpc

• Lineage Restriction Analysis:

  • FOXD3 is co-expressed with Sox10 (an NCSC and glial marker) at the periphery of vagal and trunk dorsal root ganglia (DRG) at 10.5 dpc

  • FOXD3 and p75 are co-expressed in neural crest at the developing esophagus, trachea, and cranial ganglia levels at 10.5-11.5 dpc

• Fate Mapping Experiments:

  • Combine FOXD3 antibody staining with lineage-tracing reporter systems (e.g., using R26R-YFP reporter strains) to track cell fate decisions

  • Research shows FOXD3 downregulation in cardiac NC before entry into the outflow tract and in cranial NC-derived mesenchyme

Research Findings:
Studies have demonstrated that FOXD3 is required to repress myofibroblast differentiation and maintain NCSCs in an undifferentiated state. Loss of FOXD3 results in:

• Biasing of multipotent neural crest progenitors toward a mesenchymal fate

• Significant reduction in the formation of multipotent (neural/glial/myofibroblast) colonies in clonal culture assays

• Increased incidence of myofibroblast-only colonies with minimal impact on neural-only or glial-only colonies

These findings underscore FOXD3's role in maintaining neural crest multipotency and preventing premature differentiation toward mesenchymal lineages .

What role does FOXD3 play in heterochromatin regulation and how can antibodies help investigate this function?

FOXD3 has been identified as a regulator of heterochromatin-mediated repression of repeat elements in stem cells, and antibodies are crucial for elucidating this function.

Molecular Mechanisms:
Research using FOXD3 antibodies in ChIP assays has revealed that:

• FOXD3 binds to and represses murine endogenous retrovirus L (MERVL) and major satellite repeats (MSRs) in mouse embryonic stem cells

• FOXD3 represses these elements by recruiting the heterochromatin histone methyltransferase SUV39H1

• This recruitment establishes the repressive H3K9me3 mark at target sites

Experimental Approaches:

• Binding Site Identification:

  • Electrophoretic Mobility Shift Assays (EMSA) with recombinant GST-FOXD3 confirm direct binding to DNA oligonucleotides representing MERVL and MSR containing FOXD3-binding sites

  • ChIP-Seq and ChIP-qPCR with FOXD3 antibodies demonstrate in vivo enrichment over MERVL-LTR, MERVL-int, and MSR, but not over L1MdA regions

• Functional Analysis:

  • Knockout/knockdown studies show that FOXD3 depletion leads to significant de-repression of MERVL and MSRs

  • In control cells, less than 1% express MERVL, while this increases to up to 21% in Foxd3 KO cells

  • Gene expression analysis in Foxd3 KO cells showed upregulation of 858 genes and downregulation of 413 genes, indicating FOXD3's primarily repressive role

• Protein Interaction Studies:

  • FOXD3 antibodies can be used in co-immunoprecipitation assays to detect FOXD3 interaction with SUV39H1 and other chromatin modifiers

  • ChIP-reChIP experiments can determine co-occupancy of FOXD3 and SUV39H1 at target loci

These findings highlight FOXD3's role in maintaining proper chromatin state in embryonic stem cells, preventing inappropriate expression of repetitive elements that could affect cellular identity.

How is FOXD3 expression altered in cancer and what methodological approaches with antibodies can assess its potential as a cancer biomarker?

Research has identified significant alterations in FOXD3 expression in cancer contexts, particularly in breast cancer. FOXD3 antibodies are essential tools for investigating these changes and their implications.

Expression Patterns in Cancer:

• FOXD3 is significantly downregulated in metastatic breast cancer compared to non-metastatic breast cancer tissues

• Western blot analysis confirms lower FOXD3 protein expression in metastatic protein lysates compared to non-metastatic lysates

• In established breast cancer cell lines, FOXD3 expression is much lower in metastatic MDA-MB-231 cells than in non-metastatic MCF-7 cells

• FOXD3 expression is significantly lower in triple-negative breast cancer (TNBC) tissues compared to non-TNBC invasive ductal breast cancer tissues

Methodological Approaches:

• Tissue Microarray Analysis:

  • Use FOXD3 antibodies for immunohistochemical staining of cancer tissue microarrays

  • Scoring systems based on staining intensity and percentage of positive cells

  • Correlation with clinicopathological parameters and patient outcomes

• Cell Line Models:

  • Western blot analysis of FOXD3 expression across non-metastatic (e.g., MCF-7) and metastatic (e.g., MDA-MB-231) cell lines

  • Functional studies involving FOXD3 overexpression or knockdown to determine effects on cancer cell phenotypes

• Correlation with Proliferation Markers:

  • Research indicates no significant correlation between FOXD3 expression and Ki-67 staining (p>0.05), suggesting FOXD3's role is not directly related to proliferation in breast cancer

• Expression in Specific Cancer Subtypes:

  • Comparison of FOXD3 expression between different molecular subtypes of breast cancer (TNBC vs. non-TNBC)

  • Potential correlation with hormone receptor status and other molecular markers

These findings suggest FOXD3's potential role as a biomarker for metastatic progression in breast cancer, with its downregulation potentially contributing to increased metastatic potential.

What are common challenges when using FOXD3 antibodies and how can they be addressed?

Researchers often encounter several challenges when working with FOXD3 antibodies. Here are evidence-based solutions to common problems:

High Background in Immunostaining:

• Problem: Non-specific binding, particularly in mouse tissues when using mouse-derived antibodies.

• Solution: Use specialized mouse-on-mouse blocking reagents (e.g., catalog numbers PK-2200-NB and MP-2400-NB) . Increase blocking time/concentration and optimize antibody dilutions.

Multiple Bands in Western Blots:

• Problem: Detection of non-specific bands or degradation products.

• Solution: Use freshly prepared samples with protease inhibitors. Optimize antibody dilution (typically 1:500-1:2000 for Western blots) . Validate with positive controls like FOXD3-transfected HEK293 cells that show the expected band at ~47-60 kDa .

Weak or No Signal:

• Problem: Low abundance of FOXD3 in certain tissues or cell types.

• Solution: Use nuclear extraction protocols to concentrate nuclear proteins. Consider signal amplification methods like tyramide signal amplification for IHC/IF. Verify expression levels in your sample type before proceeding.

Cross-Reactivity Issues:

• Problem: Antibody binds to proteins other than FOXD3.

• Solution: Compare multiple antibodies from different sources. Validate specificity using FOXD3 knockout/knockdown controls. Use peptide competition assays to confirm binding specificity.

Variable Results Across Applications:

• Problem: An antibody works well for one application but poorly for others.

• Solution: Select application-specific validated antibodies. Not all antibodies perform equally across applications - check validation data for your specific application .

How should researchers interpret contradictory results when using different FOXD3 antibodies?

When facing contradictory results with different FOXD3 antibodies, a systematic approach is necessary:

Validation Strategy:

• Compare Epitope Recognition:

  • Antibodies targeting different epitopes may give different results

  • N-terminal antibodies (aa 1-140) are common but may detect different isoforms than those targeting C-terminal regions

• Assess Antibody Specificity:

  • Perform side-by-side validation with multiple antibodies

  • Use genetic controls (FOXD3 knockout/knockdown) to determine true specificity

  • Conduct peptide competition assays with the immunizing peptide

• Consider Context-Dependent Expression:

  • FOXD3 expression varies significantly across developmental stages and cell types

  • Expression may be transient during development (present in pre-migratory NC at 8.0 dpc but downregulated in ventralmost NC cells by 9.0 dpc)

Interpretation Guidelines:

• When results contradict published literature: Verify antibody lot, experimental conditions, and cell/tissue type; FOXD3 function can be context-dependent

• When multiple antibodies give different results: The antibody recognizing the known molecular weight (47-60 kDa) with appropriate controls is likely more reliable

• When expression patterns differ from expectations: Consider developmental timing, as FOXD3 expression is highly dynamic during development

Documentation Practice:
Always document the specific antibody used (catalog number, lot, dilution) and validation controls in publications to facilitate reproducibility and proper interpretation of results.

How can FOXD3 antibodies contribute to understanding the role of FOXD3 in immune regulation?

Recent research has uncovered FOXD3's unexpected role in immune regulation, particularly in B cell function, opening new applications for FOXD3 antibodies.

Regulatory B Cells and IL-10 Production:
Research has demonstrated that FOXD3 suppresses interleukin-10 (IL-10) expression in B cells, affecting the development of regulatory B cells (Breg cells) . FOXD3 antibodies can be employed to investigate:

• Mechanism of IL-10 Suppression:

  • ChIP assays using FOXD3 antibodies show direct binding of FOXD3 to the IL-10 promoter in B cells

  • Knockdown of FOXD3 using shRNA promotes Breg cell production by upregulating IL-10 expression

• Expression in Autoimmune Contexts:

  • Up-regulated FOXD3 expression is negatively associated with IL-10+ Breg cells in lupus-prone MRL/lpr mice

  • FOXD3 antibodies can be used to track expression levels in various autoimmune disease models

• B Cell-Specific Functions:

  • Western blot analysis using FOXD3 antibodies can quantify expression levels in B cells under different stimulation conditions (e.g., LPS, PMA, ionomycin)

  • Flow cytometry with FOXD3 antibodies can identify FOXD3-expressing B cell subpopulations

Methodological Approach for B Cell Studies:

• Isolate B220+ B cells and culture with LPS (1 μg/ml) for stimulation

• Use validated FOXD3 antibodies for Western blot analysis (dilution 1:20,000 in TBS-T with 5% BSA)

• Employ FOXD3-specific shRNA for knockdown studies to confirm functional effects

These findings suggest potential therapeutic applications by regulating FOXD3 expression to modulate Breg cell production for treating autoimmune diseases .

What are the future prospects for using FOXD3 antibodies in regenerative medicine and stem cell research?

FOXD3 antibodies hold significant promise for advancing regenerative medicine and stem cell research due to FOXD3's critical roles in pluripotency maintenance and lineage decisions.

Pluripotent Stem Cell Quality Assessment:

• FOXD3 is required for maintenance of pluripotent cells in pre-implantation and peri-implantation stages of embryogenesis

• FOXD3 antibodies can be used to assess the quality and pluripotency status of embryonic stem cells

• Immunostaining of human embryonic stem cells (e.g., BG01V) shows FOXD3 localization in nuclei and cytoplasm

Neural Crest Stem Cell Identification and Isolation:

• FOXD3 expression identifies multipotent neural crest stem cells

• FOXD3 antibodies combined with other markers (Sox10, p75) can help purify NCSCs for regenerative applications

• Co-expression analysis shows that at 10.5 dpc, FOXD3 is co-expressed with Sox10 at the periphery of dorsal root ganglia

Lineage Specification Monitoring:

• FOXD3 mediates a fate restriction choice for multipotent neural crest progenitors, with loss of FOXD3 biasing neural crest toward a mesenchymal fate

• FOXD3 antibodies can track this transition during differentiation protocols

• Individual control neural crest cells typically form tightly packed colonies whereas FOXD3-deficient colonies consist of loosely-packed fibroblast-like cells

Future Technological Applications:

• Antibody-based Sorting: Development of FOXD3 antibody-based cell sorting strategies for isolating specific progenitor populations

• Live Imaging: Creation of non-disruptive FOXD3 antibody-based probes for live cell imaging of lineage decisions

• Bioprinting: Incorporation of FOXD3 expression assessment in quality control for bioprinted tissues containing neural crest derivatives

As regenerative medicine advances toward clinical applications, FOXD3 antibodies will likely become essential tools for quality control and monitoring of stem cell-derived products.

Comprehensive reporting of antibody details is essential for experimental reproducibility. Based on best practices from FOXD3 research literature, authors should include:

Primary Antibody Documentation:

• Complete catalog information: manufacturer, catalog number, clone designation (for monoclonal antibodies)

• Host species and antibody type (monoclonal/polyclonal)

• Lot number (particularly for polyclonal antibodies)

• RRID (Research Resource Identifier) when available

• Epitope/immunogen information (e.g., "Recombinant Human FOXD3 protein aa 1-140")

Validation Evidence:

• Citation of previous validation studies or inclusion of validation data

• Description of controls used (positive, negative, peptide competition)

• For novel applications, include complete validation data

Application-Specific Details:

• Exact dilution used for each application

• Incubation conditions (time, temperature)

• For IHC/IF: antigen retrieval method, blocking procedure, detection system

• For WB: protein amount loaded, gel percentage, transfer method

• For ChIP: chromatin preparation method, antibody amount, washing conditions

Example of Proper Documentation:
"FOXD3 was detected using rabbit polyclonal antibody to human FOXD3 (Company X, Cat# Y123, RRID:AB_123456, Lot# Z789) raised against recombinant human FOXD3 (aa 1-140). The antibody was used at 1:500 dilution for Western blot and 1:200 for immunohistochemistry. Specificity was confirmed using FOXD3-transfected HEK293 cells as positive control and mock-transfected cells as negative control."

Adherence to these reporting standards will significantly enhance reproducibility in FOXD3 research and facilitate meta-analyses across studies.