HOXD3 Antibody, FITC conjugated

What is HOXD3 and what role does it play in biological systems?

HOXD3 (Homeobox D3) is a sequence-specific transcription factor that functions as part of a developmental regulatory system providing cells with specific positional identities on the anterior-posterior axis . Recent research has demonstrated that HOXD3 promotes migration and angiogenesis in hepatocellular carcinoma (HCC) by targeting the promoter region of CCR6 and inducing its transcription . The protein has a molecular weight of approximately 34-45 kDa and is expressed in several tissue types. HOXD3 has been implicated in various biological processes including cell migration, invasion, and angiogenesis, making it a significant target for research in developmental biology and cancer progression studies.

What are the key specifications of commercially available HOXD3 Antibody, FITC conjugated?

The HOXD3 Antibody, FITC conjugated (e.g., ABIN7155772) is a polyclonal antibody raised in rabbit that targets the amino acid sequence 263-346 of human HOXD3 protein . The antibody has undergone Protein G purification with >95% purity and uses recombinant Human Homeobox protein Hox-D3 protein (263-346AA) as the immunogen . The reagent is typically supplied in a liquid formulation containing preservatives such as Proclin 300 (0.03%), with 50% glycerol and 0.01M PBS at pH 7.4 . Its reactivity is primarily with human samples, and it is specifically designed for research applications, not diagnostic or therapeutic purposes .

How does the epitope selection (AA 263-346) for the FITC-conjugated HOXD3 antibody influence its applications?

The epitope selection targeting amino acids 263-346 of HOXD3 covers a critical functional region of the protein. This region appears to be relatively conserved in humans but may differ across species, explaining the antibody's specific reactivity to human samples . The selection of this particular epitope offers advantages for studying HOXD3's role in transcriptional regulation and protein-protein interactions, as this region may be involved in DNA binding or regulatory functions. When designing experiments, researchers should consider that this epitope selection might influence the antibody's ability to recognize HOXD3 in different conformational states or protein complexes. For cross-species studies, researchers should verify sequence homology in the epitope region before attempting to use this antibody in non-human models.

What are the optimal fixation and permeabilization protocols for HOXD3 antibody in immunofluorescence studies?

For optimal results with HOXD3 antibody in immunofluorescence applications, paraformaldehyde fixation (4%) followed by Triton X-100 permeabilization (0.1-0.5%) has shown good results . The following protocol is recommended:

• Fix cells on coverslips with 4% paraformaldehyde for 15-20 minutes at room temperature

• Wash 3x with PBS (5 minutes each)

• Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes

• Block with 5% normal serum (matched to secondary antibody host) in PBS with 0.1% Triton X-100 for 1 hour

• Incubate with HOXD3 antibody, FITC conjugated at 2-4 μg/mL in blocking buffer overnight at 4°C

• Wash 3x with PBS (5 minutes each)

• Mount with anti-fade medium containing DAPI

For tissues, extend fixation time appropriately and consider using a lower concentration of Triton X-100 (0.1%) during permeabilization to preserve tissue morphology while maintaining adequate antibody accessibility to the nuclear target.

How should researchers design co-localization studies involving FITC-conjugated HOXD3 antibody?

When designing co-localization studies with FITC-conjugated HOXD3 antibody, consider the following methodological approach:

• Fluorophore selection: Since HOXD3 antibody is conjugated to FITC (green fluorescence), select compatible fluorophores for co-staining that have minimal spectral overlap, such as TRITC/Cy3 (red) or Cy5/Alexa 647 (far-red).

• Nuclear targeting considerations: Since HOXD3 is a transcription factor with nuclear localization, pair it with appropriate nuclear or subnuclear markers such as:

  • RNA Polymerase II for transcription factories

  • Specific histone modifications (H3K4me3, H3K27ac) for active promoters

  • CCR6 or other downstream targets to confirm regulatory relationships

• Controls: Include single-stained controls for each fluorophore to establish proper exposure settings and confirm absence of bleed-through.

• Image acquisition: Utilize sequential scanning on confocal microscopy rather than simultaneous acquisition to minimize cross-talk between channels.

• Quantification: Employ colocalization analysis software that calculates Pearson's correlation coefficient or Manders' overlap coefficient to quantitatively assess spatial relationships.

What validation steps should be performed before using HOXD3 antibody in critical experiments?

Before employing the HOXD3 antibody, FITC conjugated in critical experiments, researchers should undertake the following validation steps:

• Positive and negative controls:

  • Positive: Cell lines known to express HOXD3 (such as HepG2 for hepatocellular studies)

  • Negative: Cell lines with minimal HOXD3 expression or HOXD3 knockout/knockdown cells

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide (HOXD3 aa 263-346) before application to samples, which should abolish specific staining.

• Cross-reactivity assessment: Test the antibody on samples from other species if planning cross-species studies, despite the antibody being primarily validated for human reactivity .

• Western blot validation: Though the FITC-conjugated antibody is primarily for immunofluorescence, testing an unconjugated version from the same clone in Western blot can confirm specificity at the expected molecular weight (approximately 34-45 kDa) .

• Comparison with alternative antibodies: Compare staining patterns with other validated HOXD3 antibodies targeting different epitopes to confirm localization pattern.

What are the common issues with photobleaching of FITC-conjugated antibodies and how can they be mitigated?

FITC conjugates are particularly susceptible to photobleaching, which can significantly impact experimental outcomes. Here are the common issues and mitigation strategies:

Common issues:

• Rapid signal fading during imaging acquisition

• Inconsistent signal intensity between slides/samples

• Poor signal-to-noise ratio in fixed samples stored for extended periods

Mitigation strategies:

• Anti-fade reagents: Mount samples using specialized anti-fade mounting media containing anti-oxidants and radical scavengers.

• Imaging parameters optimization:

  • Reduce exposure time and laser/light intensity

  • Increase gain/sensitivity if possible

  • Utilize frame averaging to improve signal-to-noise ratio

  • Capture FITC channel images first in multi-channel acquisition

• Sample preparation:

  • Shield samples from light during all processing steps

  • Store slides at -20°C in the dark

  • Consider using vacuum-sealed containers for long-term storage

• Alternative approaches:

  • For critical experiments requiring multiple viewing sessions, consider using more photostable fluorophores (Alexa 488 conjugated antibodies instead of FITC)

  • In live cell imaging applications, consider using lower antibody concentrations (1-2 μg/mL) and supplementing media with vitamin C (ascorbic acid, 100 μM) as an antioxidant

How can researchers optimize signal-to-noise ratio when working with HOXD3 antibody in cells with low expression levels?

Optimizing signal-to-noise ratio for HOXD3 detection in low-expression systems requires a methodical approach:

• Sample preparation optimization:

  • Extend primary antibody incubation to overnight at 4°C

  • Use a higher antibody concentration (4-6 μg/mL) for low-expressing samples

  • Optimize fixation time to preserve epitope accessibility (12-15 minutes may be optimal)

  • Consider mild antigen retrieval methods if appropriate for your sample type

• Background reduction strategies:

  • Increase blocking time to 2 hours using 5-10% serum with 0.3% Triton X-100

  • Add 0.1-0.2% BSA to antibody dilution buffer

  • Include 0.05% Tween-20 in wash buffers

  • Perform additional wash steps (5-6 washes rather than standard 3)

• Detection enhancement:

  • Use a high-sensitivity detection system if working with an unconjugated primary

  • Employ image acquisition settings optimized for low-signal detection (longer exposure, higher gain)

  • Utilize deconvolution software for image processing

  • Consider signal amplification methods such as tyramide signal amplification if compatible with experimental design

• Positive controls: Always include a positive control sample with known high HOXD3 expression (such as HepG2 cells) to confirm antibody functionality.

What are the best storage conditions to maintain FITC-conjugated HOXD3 antibody activity for extended periods?

To maintain optimal activity of FITC-conjugated HOXD3 antibody during storage:

• Short-term storage (up to 1 month):

  • Store at 4°C protected from light

  • Add sodium azide to a final concentration of 0.02% if not already present in the formulation

  • Avoid repeated freeze-thaw cycles

• Long-term storage (beyond 1 month):

  • Store at -20°C in the dark

  • Aliquot into small volumes (5-20 μL) before freezing to avoid repeated freeze-thaw cycles

  • Use dark-colored tubes or wrap in aluminum foil to protect from light

  • Include a desiccant if possible to prevent moisture condensation during temperature changes

• Working solution preparation:

  • Thaw aliquots rapidly at room temperature

  • Centrifuge briefly before opening to collect all liquid

  • Prepare fresh working dilutions on the day of the experiment

  • Do not refreeze diluted antibody

• Stability monitoring:

  • Test activity periodically on positive control samples

  • Note any decrease in staining intensity or increase in background as indicators of degradation

  • Typical shelf-life for properly stored FITC conjugates is 6-12 months

How can HOXD3 antibody, FITC conjugated be utilized in ChIP-sequencing experiments to identify genomic binding sites?

Although FITC-conjugated antibodies are not typically used directly in ChIP-seq, researchers can adapt protocols using the same antibody clone without FITC conjugation for ChIP applications. Based on recent findings about HOXD3's role in transcriptional regulation , a ChIP-seq approach would involve:

• Experimental design considerations:

  • Focus on regulatory regions of CCR6, Med15, and CREBBP genes identified as HOXD3 targets

  • Include appropriate positive controls (known HOXD3 binding sites) and negative controls (non-bound regions)

  • Consider using a dual crosslinking approach (DSG followed by formaldehyde) for optimal capture of transcription factor complexes

• Protocol modifications for HOXD3:

  • Use unconjugated version of the same antibody clone

  • Optimize chromatin fragmentation to 200-300bp

  • Increase antibody concentration (4-5 μg per ChIP reaction)

  • Extend incubation time to ensure efficient immunoprecipitation

• Data analysis focus:

  • Search for enrichment near promoter regions of genes involved in cell migration, invasion, and angiogenesis

  • Analyze for common DNA binding motifs in HOXD3-bound regions

  • Compare binding profiles in normal versus cancer cell lines to identify differential regulatory patterns

  • Integrate with RNA-seq data to correlate binding with transcriptional outcomes

• Validation of ChIP-seq findings:

  • Confirm selected binding sites using ChIP-qPCR

  • Validate functional relevance through reporter assays or CRISPR-mediated deletion of binding sites

What approaches can researchers use to study the HOXD3-CCR6 regulatory axis in cancer progression models?

Based on the identification of the HOXD3-CCR6 regulatory axis in hepatocellular carcinoma , researchers can employ several approaches to further investigate this pathway:

• Co-expression analysis in patient samples:

  • Perform dual immunofluorescence using HOXD3 antibody, FITC conjugated and anti-CCR6 antibody (with compatible fluorophore)

  • Quantify correlation between HOXD3 and CCR6 expression levels

  • Correlate expression patterns with clinical parameters and patient outcomes

• Functional validation in cell models:

  • Generate HOXD3 knockout/knockdown and overexpression models

  • Assess changes in CCR6 expression using qRT-PCR, Western blot, and immunofluorescence

  • Perform migration, invasion, and angiogenesis assays to assess functional outcomes

  • Rescue experiments by introducing CCR6 expression in HOXD3-deficient cells

• Mechanistic studies of transcriptional regulation:

  • Use luciferase reporter assays with CCR6 promoter constructs to validate direct regulation

  • Perform site-directed mutagenesis of predicted HOXD3 binding sites in the CCR6 promoter

  • Analyze chromatin accessibility changes (ATAC-seq) at the CCR6 locus upon HOXD3 modulation

  • Investigate cofactor requirements through co-immunoprecipitation studies

• In vivo validation:

  • Develop xenograft models with HOXD3-modulated cancer cells

  • Assess tumor growth, metastasis, and angiogenesis

  • Analyze exosome-mediated transfer of CCR6 to endothelial cells in tumor microenvironment

  • Test therapeutic approaches targeting this regulatory axis

How can HOXD3 antibody be employed in studying exosome-mediated intercellular communication in cancer?

Recent research has shown that CCR6, regulated by HOXD3, can be transported to endothelial cells by exosomes in the tumor microenvironment . To study this process:

• Exosome isolation and characterization:

  • Isolate exosomes from culture media of HOXD3-overexpressing and control cells

  • Characterize exosomes by nanoparticle tracking analysis, electron microscopy, and Western blotting for exosomal markers

  • Analyze exosomal content for CCR6 protein and mRNA levels

• Exosome labeling and trafficking studies:

  • Label isolated exosomes with fluorescent membrane dyes

  • Track uptake by recipient endothelial cells using live-cell imaging

  • Co-stain for HOXD3 (using FITC-conjugated antibody) and CCR6 in recipient cells

  • Assess colocalization and temporal dynamics of protein expression post-exosome uptake

• Functional analysis of exosome-mediated effects:

  • Perform tube formation assays with endothelial cells treated with exosomes

  • Analyze migration, invasion, and angiogenic potential

  • Block exosome uptake using various inhibitors to confirm specificity

  • Deplete specific exosomal components to identify key mediators

• In vivo exosome tracking:

  • Label exosomes with near-infrared dyes for in vivo imaging

  • Track biodistribution in tumor-bearing animals

  • Analyze tumor sections for exosome uptake by endothelial cells

  • Correlate with markers of angiogenesis and tumor progression

How should researchers quantify HOXD3 nuclear localization signal in immunofluorescence studies?

For accurate quantification of HOXD3 nuclear localization:

• Image acquisition parameters:

  • Capture images at 40-63x magnification using confocal microscopy

  • Maintain consistent exposure settings across all samples

  • Collect z-stacks (0.5-1 μm steps) to capture the full nuclear volume

  • Include DAPI counterstain for nuclear identification

• Quantification methodologies:

  • Nuclear/cytoplasmic ratio analysis:

    • Define nuclear regions based on DAPI staining

    • Measure mean FITC intensity in nuclear and cytoplasmic regions

    • Calculate nuclear/cytoplasmic ratio for each cell

    • Analyze at least 50-100 cells per condition

  • Nuclear intensity distribution:

    • Generate intensity histograms of nuclear HOXD3-FITC signal

    • Compare distribution patterns between experimental conditions

    • Assess for shifts in population distributions rather than just mean values

  • Subnuclear localization patterns:

    • Classify nuclear staining patterns (diffuse, punctate, peripheral)

    • Quantify number and intensity of nuclear foci if present

    • Correlate patterns with functional states (active vs. inactive)

• Statistical analysis:

  • Use appropriate statistical tests based on data distribution

  • For comparing multiple conditions, employ ANOVA with post-hoc tests

  • Consider hierarchical analysis to account for cell-to-cell variability within samples

  • Present data as box plots or violin plots to show distribution characteristics

What controls and validation steps are necessary when analyzing HOXD3 expression in clinical samples?

When analyzing HOXD3 expression in clinical samples:

• Essential controls:

  • Positive tissue controls: Include samples known to express HOXD3 (developmental tissues or specific cancer types)

  • Negative controls: Include tissues known to have minimal HOXD3 expression

  • Technical controls:

    • Omit primary antibody (secondary-only control)

    • Include isotype control at equivalent concentration

    • Use blocking peptide competition to confirm specificity

• Sample processing validation:

  • Verify consistent fixation quality across samples

  • Confirm nuclear antigen preservation using control nuclear markers

  • Standardize staining protocols, including antigen retrieval methods

  • Process all samples in the same batch when possible to minimize technical variation

• Analytical validation:

  • Implement blinded scoring/quantification by multiple observers

  • Establish clear scoring criteria before analysis

  • Use digital image analysis when possible to reduce subjective interpretation

  • Validate findings with orthogonal methods (qRT-PCR, Western blot) when feasible

  • Correlate HOXD3 expression with known downstream targets (e.g., CCR6)

• Clinical data integration:

  • Stratify analyses based on relevant clinical parameters

  • Control for potential confounding variables

  • Consider multiple hypothesis testing correction for correlative analyses

  • Validate findings in independent patient cohorts when possible

How can researchers distinguish between specific and non-specific signals when using HOXD3 antibody in complex tissue samples?

Distinguishing specific from non-specific signals in complex tissues requires rigorous methodology:

• Technical approaches to enhance specificity:

  • Titrate antibody concentration to determine optimal signal-to-noise ratio

  • Extend blocking time (2-3 hours) with 5-10% normal serum from the same species as the secondary antibody

  • Add 0.1-0.3% Triton X-100 to blocking solution to reduce hydrophobic interactions

  • Increase wash duration and number of washes

  • Consider using specialized blockers for endogenous biotin or peroxidase if relevant

• Validation controls:

  • Absorption controls: Pre-incubate antibody with immunizing peptide

  • Knockout validation: When available, use HOXD3 knockout tissue as negative control

  • Antibody comparison: Test multiple antibodies against different HOXD3 epitopes

  • RNA-protein correlation: Compare antibody staining pattern with in situ hybridization for HOXD3 mRNA

• Pattern recognition for specific HOXD3 staining:

  • Specific staining should show predominantly nuclear localization

  • Signal intensity should correlate with known expression patterns

  • Staining should show biological gradient across tissue regions where relevant

  • Cellular staining should be consistent with known subcellular localization

• Quantitative approach:

  • Plot signal intensity histograms comparing specific regions of interest vs. background

  • Determine threshold values based on negative controls

  • Consider autofluorescence spectra and implement appropriate corrections

  • Use spectral unmixing for complex tissues with high autofluorescence

What are the advantages and limitations of FITC-conjugated HOXD3 antibody compared to other detection methodologies?

How do researchers reconcile discrepancies in HOXD3 localization or expression patterns between different detection methods?

When faced with discrepancies in HOXD3 detection between methods, researchers should follow this analytical framework:

• Methodological considerations:

  • Epitope availability: Different fixation methods may mask or expose different epitopes

  • Antibody specificity: Confirm whether antibodies target different regions of HOXD3

  • Detection thresholds: Assess sensitivity differences between methods

  • Sample preparation variations: Standardize protocols to eliminate technical variables

• Biological interpretation:

  • Protein vs. mRNA discrepancies: Consider post-transcriptional regulation

  • Differential splicing: Verify whether antibodies detect different isoforms

  • Protein modification state: Assess whether antibodies are sensitive to phosphorylation or other modifications

  • Protein complexes: Determine if protein interactions mask epitopes in native conditions

• Resolution strategies:

  • Orthogonal validation: Implement a third, independent method

  • Controlled manipulation: Use overexpression or knockdown to confirm antibody specificity

  • Domain-specific analysis: Use multiple antibodies targeting different regions

  • Subcellular fractionation: Biochemically separate cellular compartments before analysis

  • Mass spectrometry validation: Confirm protein identity in immunoprecipitated samples

• Consensus approach:

  • Weight evidence based on methodological rigor

  • Consider biological plausibility of each result

  • Acknowledge limitations in publication and discussion

  • Present multiple lines of evidence rather than selecting only consistent results

What emerging technologies might complement or replace traditional antibody-based detection of HOXD3 in research settings?

Several cutting-edge technologies are poised to complement or potentially replace traditional antibody-based detection of HOXD3:

• CRISPR-based tagging:

  • CRISPR knock-in of fluorescent proteins to endogenous HOXD3

  • Advantages: Endogenous expression levels, live-cell imaging compatibility

  • Limitations: Resource-intensive, potential functional interference, cell/organism-specific

• Proximity ligation assays (PLA):

  • Detection of protein-protein interactions involving HOXD3

  • Advantages: Single-molecule sensitivity, visualization of specific interaction partners

  • Applications: Studying HOXD3 interactions with CREBBP/Med15 complex

• Mass cytometry (CyTOF):

  • Metal-tagged antibodies for high-dimensional single-cell analysis

  • Advantages: No spectral overlap issues, simultaneous measurement of >40 parameters

  • Applications: Comprehensive characterization of HOXD3 in heterogeneous tissues

• Spatial transcriptomics:

  • In situ sequencing approaches to visualize HOXD3 mRNA in spatial context

  • Advantages: Transcriptome-wide perspective, preservation of tissue architecture

  • Limitations: Protein-level regulation not captured

• Aptamer-based detection:

  • Synthetic nucleic acids selected for specific binding to HOXD3 protein

  • Advantages: Highly specific, reproducible, not animal-derived

  • Status: Emerging technology, not yet widely available for most targets

• Single-cell proteomics:

  • Mass spectrometry-based approaches for single-cell protein quantification

  • Advantages: Unbiased detection, isoform differentiation

  • Limitations: Currently limited sensitivity for low-abundance transcription factors

• Nanobody technology:

  • Single-domain antibodies derived from camelid immune systems

  • Advantages: Smaller size, better tissue penetration, reduced immunogenicity

  • Applications: Super-resolution microscopy of nuclear factors