HOXB3 Antibody, FITC conjugated

What is the optimal application range for HOXB3 antibody, FITC conjugated?

HOXB3 antibodies conjugated with FITC are primarily used for:

• Immunohistochemistry (IHC) on frozen and paraffin-embedded tissues

• Immunofluorescence (IF) for cellular localization studies

• Flow cytometry for quantitative analysis of cell populations

• ELISA for protein quantification

The optimal dilution range varies by application:

• For IHC: 1:50-1:200

• For IF: 1:100-1:500

• For flow cytometry: 1:20-1:100

• For ELISA: 1:1000-1:3000

Most HOXB3 antibodies recognize epitopes within the C-terminal region (amino acids 315-423 in humans), which contains the DNA-binding homeodomain, making them suitable for detecting both nuclear localization and DNA-binding activities .

What are the species cross-reactivity profiles of FITC-conjugated HOXB3 antibodies?

Species reactivity depends on sequence conservation and the specific epitope targeted:

While many manufacturers claim multi-species reactivity, it's important to note that actual cross-reactivity should be experimentally validated. For instance, HOXB3 antibodies from suppliers like Qtonics specifically note human reactivity , while others from G-Biosciences claim reactivity with human, mouse and rat samples .

For novel cross-species applications, researchers should consider conducting preliminary validation experiments with appropriate positive and negative controls .

How does the FITC conjugation process affect HOXB3 antibody performance?

FITC conjugation occurs via primary amines (lysine residues) on the antibody:

• Optimal conjugation typically involves 3-6 FITC molecules per antibody molecule

• Higher FITC:antibody ratios (>6:1) can cause:

  • Solubility problems

  • Internal quenching effects

  • Reduced fluorescence brightness

  • Altered binding kinetics

FITC-conjugated antibodies have specific spectral properties:

• Excitation maximum: 488 nm (argon laser)

• Emission maximum: 530 nm

• Quantum yield: 0.85 in optimal pH (7.4-8.0)

• pH sensitivity: fluorescence intensity decreases at pH <7.0

To optimize performance, manufacturers typically prepare multiple conjugation ratios and select the optimal preparation based on brightness and background testing. Researchers should:

• Store antibodies according to manufacturer guidelines (typically -20°C)

• Protect from light exposure

• Avoid repeated freeze-thaw cycles

• Use stabilizing proteins (BSA) in dilution buffers

What are the most effective fixation and permeabilization methods for HOXB3 immunofluorescence using FITC-conjugated antibodies?

Optimal methods depend on subcellular localization and experimental goals:

For nuclear HOXB3 detection (most common):

• Fixation options:

  • 4% paraformaldehyde (15-20 min at RT) - Preserves structure while maintaining antigenicity

  • 80% methanol (5 min) followed by permeabilization with 0.1% PBS-Tween (20 min) - Effective for nuclear proteins

• Permeabilization options:

  • 0.1-0.5% Triton X-100 in PBS (10 min) - Provides good nuclear access

  • 0.1% PBS-Tween (20 min) - Gentler option

• Blocking recommendation:

  • 1× PBS containing 10% normal serum and 0.3M glycine (30-60 min) to block non-specific interactions

Important considerations:

• Over-fixation can mask epitopes

• HOXB3 detection in hematopoietic cells may require specialized fixation protocols

• For dual staining applications, fixation must be compatible with all target proteins

• Inclusion of phosphatase and protease inhibitors during sample preparation improves detection of labile phosphorylation states

For validation of staining patterns, compare results with published nuclear localization patterns of HOXB3 in cell types where it shows transcriptional activity .

How can I overcome autofluorescence when using FITC-conjugated HOXB3 antibodies in tissue sections?

Tissue autofluorescence poses significant challenges for FITC detection, particularly in:

• Tissues rich in elastin and collagen

• Samples with lipofuscin

• Formalin-fixed tissues

Effective strategies include:

• Pretreatment methods:

  • 0.1-1% sodium borohydride in PBS (10 min)

  • 0.1-0.3% Sudan Black B in 70% ethanol (20 min)

  • 10 mM cupric sulfate in 50 mM ammonium acetate buffer (pH 5.0)

• Imaging approaches:

  • Spectral unmixing on confocal microscopes

  • Time-gated detection (FITC has longer fluorescence lifetime than autofluorescence)

  • Linear unmixing algorithms during image processing

• Alternative detection:

  • Consider using secondary detection systems with longer wavelength fluorophores

  • Use nuclear counterstains that don't interfere with FITC emission

Quantitative comparison approach:
Set up a control experiment with:

• Primary + FITC-conjugated secondary

• Isotype control + FITC-conjugated secondary

• Autofluorescence only (no antibody)

Subtract the mean fluorescence intensity of controls from experimental samples for accurate quantification .

What controls are essential for validating specificity of FITC-conjugated HOXB3 antibodies?

Rigorous validation requires multiple controls:

Negative controls:

• Isotype control: Use matched isotype antibody (typically rabbit IgG) with FITC conjugation at same concentration

• Peptide competition: Pre-incubate antibody with immunizing peptide (10-100× excess)

• Null/knockout model: Test in HOXB3-null cells, if available

• siRNA knockdown: Compare staining in cells with HOXB3 knockdown vs. control

Positive controls:

• Western blot validation: Confirm single band at expected molecular weight (~44kDa)

• Known positive tissues: Test in tissues with established HOXB3 expression (e.g., CD34+ early hematopoietic cells, certain prostate cancer lines)

• Recombinant protein expression: Test in overexpression systems

Multi-method validation:
Cross-validate results using independent techniques:

• RT-PCR to confirm mRNA expression

• RNA-seq data correlation

• Alternative antibody clones against different epitopes

Published research suggests validating HOXB3 antibody specificity against multiple HOX family members, as cross-reactivity can occur due to homeodomain conservation. Particular attention should be paid to distinguishing HOXB3 from the closely related HOXA3 and HOXB4 proteins .

How can I optimize detection of low HOXB3 expression in rare hematopoietic stem cell populations?

HOXB3 is expressed at high levels in early CD34+ lineage-negative bone marrow cells but decreases during differentiation, making detection challenging in rare populations :

Flow cytometry optimization:

• Cell enrichment strategies:

  • MACS selection for CD34+ cells prior to staining

  • Lineage depletion to enrich for primitive populations

  • Use of c-Kit+Flk-2-lineage-Sca-1+ markers to identify HSCs

• Signal amplification methods:

  • Tyramide signal amplification (TSA)

  • Multi-layer staining approaches

  • Brighter FITC conjugates with optimized F/P ratios

• Instrument settings:

  • Optimize PMT voltages specifically for FITC

  • Use compensation controls to address spectral overlap

  • Employ narrow bandpass filters for improved signal:noise

Protocol refinements for immunofluorescence:

• Extended primary antibody incubation (overnight at 4°C)

• Use of signal enhancers like ProLong Gold with antifade

• Sequential scanning in confocal microscopy

When analyzing HOXB3 in hematopoietic cells, co-staining with markers like CD34, c-Kit, and lineage markers allows identification of specific developmental stages where HOXB3 expression changes are biologically relevant .

What are the best approaches for studying HOXB3-mediated transcriptional regulation using chromatin immunoprecipitation (ChIP)?

HOXB3 functions as a sequence-specific transcription factor regulating developmental and oncogenic programs:

ChIP protocol optimization:

• Crosslinking options:

  • 1% formaldehyde (10 min at RT) for direct DNA binding

  • Dual crosslinking with 1.5mM EGS followed by formaldehyde for protein complexes

• Chromatin fragmentation:

  • Sonication to 200-500bp fragments

  • Enzymatic digestion alternatives for sensitive epitopes

• Antibody considerations:

  • FITC-conjugated antibodies can be used with anti-FITC beads

  • Non-conjugated antibodies often perform better in ChIP

  • 2-5μg antibody per ChIP reaction

Analysis of HOXB3 binding regions:

• HOXB3 binds to consensus sequence 5'-TAAT-3' within larger motifs

• Target genes include WNT pathway components and cell cycle regulators

• In prostate cancer, HOXB3 regulates super-enhancer regions

Validation approaches:

• qPCR for known targets

• ChIP-seq for genome-wide binding analysis

• Integration with RNA-seq to correlate binding with expression changes

Recent research has shown that HOXB3 transcriptionally regulates multiple WNT pathway genes and can function as a downstream transcription factor in the WNT pathway in castration-resistant prostate cancer . HOXB3 ChIP studies should consider examining the relationship between HOXB3 binding and H3K27ac marks, as they frequently co-localize at enhancer regions .

How do I design experiments to investigate HOXB3's role in hematopoietic stem cell function?

HOXB3 plays important roles in hematopoietic stem cell proliferation and differentiation :

Experimental approaches:

• Loss-of-function studies:

  • Conditional knockout models

  • shRNA/CRISPR in primary HSCs

  • Compare with related factors (HOXA3, HOXB4)

  Assessment	Readout	Expected Result
  Colony-forming assays	HPP-CFC counts	Reduced in HOXB3 knockout
  Competitive transplantation	Donor chimerism	Reduced repopulating ability
  Cell cycle analysis	Ki67/7AAD staining	Altered cycling status
  Lineage differentiation	Flow cytometry	Defects in lymphoid development

• Gain-of-function approaches:

  • Retroviral overexpression in bone marrow

  • Inducible expression systems

  • Expected outcomes: increased GMPs, decreased lymphoid development

• Cytokine response testing:

  • Measure proliferative response to SCF, TPO, Flt-3 ligand

  • Assess colony formation with GM-CSF, IL-3, G-CSF

Based on studies with related HOX genes, researchers should examine how HOXB3 expression affects:

• Hematopoietic stem cell self-renewal vs. differentiation

• Response to hematopoietic stress (5-FU, irradiation)

• Long-term vs. short-term repopulating ability

Studies of HOXB3 function should be compared with the better-characterized HOXA9 and HOXB4 genes, which have established roles in hematopoietic stem cell regulation .

What methodologies are recommended for investigating HOXB3's role in cancer progression?

Recent research has identified HOXB3 as a significant factor in cancer progression, particularly in prostate cancer :

Experimental approaches:

• Expression analysis:

  • IHC in tissue microarrays (correlate with clinical outcomes)

  • FITC-conjugated antibodies for flow cytometry/IF in patient-derived samples

  • Quantitative assessment via Western blot

• Functional studies:

  • CRISPR knockout in cancer cell lines

  • siRNA knockdown

  • Overexpression studies with wild-type vs. mutant HOXB3

• WNT pathway interaction analysis:

  • Assess effects of WNT3A stimulation on HOXB3 activity

  • Examine HOXB3 nuclear translocation upon WNT activation

  • Investigate effects of APC deficiency on HOXB3 function

• Therapeutic targeting approaches:

  • HOXB3 inhibition in APC-deficient CRPC models

  • Combination with anti-androgen therapy

  • Assessment of drug resistance mechanisms

Key methodological considerations:

• Use multiple cancer and normal cell lines

• Validate antibody specificity in each experimental system

• Employ both in vitro and in vivo models

• Include patient-derived xenografts when possible

Recent findings indicate that HOXB3 protein level is an independent risk factor for PSA progression and death in patients with metastatic CRPC. Furthermore, suppression of HOXB3 can reduce cell proliferation in APC-downregulated CRPC cells and resensitize APC-deficient CRPC xenografts to abiraterone . These findings suggest HOXB3 as a potential therapeutic target specifically in WNT-activated prostate cancers.

How can I minimize photobleaching of FITC-conjugated HOXB3 antibodies during long-duration imaging?

FITC is particularly susceptible to photobleaching compared to other fluorophores:

Preventive strategies:

• Sample preparation:

  • Use antifade mounting media containing radical scavengers

  • ProLong Gold, Vectashield, or custom solutions with p-phenylenediamine

  • Consider oxygen-scavenging systems (glucose oxidase/catalase)

• Microscopy settings:

  • Reduce excitation intensity (20-50% of maximum)

  • Minimize exposure time

  • Use neutral density filters

  • Employ shutters to limit illumination to image acquisition periods

• Advanced imaging approaches:

  • Resonant scanning in confocal microscopy

  • Light sheet microscopy for reduced photodamage

  • Spinning disk confocal for faster acquisition with less exposure

Comparative photobleaching rates:

• Image acquisition strategies:

  • Acquire FITC channels first in multi-color experiments

  • Use time-lapse intervals that balance temporal resolution with photobleaching

  • Consider deconvolution to improve signal from lower-intensity images

What is the optimal approach for dual immunolabeling when using FITC-conjugated HOXB3 antibodies?

When combining FITC-conjugated HOXB3 antibodies with other fluorophores:

Channel selection strategies:

• Optimal fluorophore combinations with FITC:

  Secondary Fluorophore	Excitation (nm)	Emission (nm)	Spectral Separation
  FITC	488	520	Reference
  TRITC/Cy3	550	570	Excellent
  APC/Cy5	650	670	Excellent
  Pacific Blue	405	455	Good
  PE	565	575	Moderate, needs compensation

• Sequential staining protocol:

  • Complete HOXB3-FITC staining first

  • Apply blocking step with excess unconjugated antibody

  • Perform second marker staining

  • This prevents potential cross-reactivity between detection systems

• Controls for dual labeling:

  • Single-color controls for compensation

  • Antibody order reversal to check for artifactual co-localization

  • Secondary-only controls to assess background

For co-localization studies of HOXB3 with nuclear markers (like transcription factors or histone modifications), confocal microscopy with careful Z-sectioning is recommended to accurately assess nuclear co-localization .

How should FITC-conjugated HOXB3 antibodies be stored to maintain optimal performance?

Proper storage is critical for maintaining antibody functionality and fluorophore integrity:

Storage recommendations:

• Temperature conditions:

  • Long-term storage: -20°C or -80°C as specified by manufacturer

  • Working aliquots: 4°C for up to 1 week

  • Avoid repeated freeze-thaw cycles (limit to <5 cycles)

• Buffer components for stability:

  • 50% Glycerol to prevent freezing damage

  • 0.01M PBS, pH 7.4 (optimal for FITC stability)

  • Preservatives like 0.03% Proclin 300 or 0.02% sodium azide

  • BSA (1-5%) for protein stabilization, unless BSA-free formulation is required

• Light protection:

  • Store in amber vials or wrapped in aluminum foil

  • Minimize exposure to light during handling

  • LED laboratory lighting causes less photobleaching than fluorescent lights

• Reconstitution guidelines:

  • Follow manufacturer's instructions precisely

  • Use sterile, high-quality water or provided diluent

  • Allow antibody to reach room temperature before opening

Storage stability data indicates that properly stored FITC conjugates typically retain >80% activity for 12 months when stored at -20°C. Antibodies stored in solution rather than lyophilized form are more susceptible to degradation over time .

How can I investigate HOXB3's interaction with the WNT signaling pathway in experimental models?

Recent research has identified HOXB3 as both a target and mediator of WNT signaling, particularly in cancer contexts :

Experimental approaches:

• Activation studies:

  • Treat cells with recombinant WNT3A

  • Use GSK3β inhibitors (CHIR99021, LiCl)

  • Express constitutively active β-catenin

  • Monitor HOXB3 localization using FITC-conjugated antibodies

• Inhibition strategies:

  • Tankyrase inhibitors (e.g., XAV939)

  • Porcupine inhibitors (e.g., LGK974)

  • dnTCF expression

  • Examine effects on HOXB3 expression and activity

• Interaction analysis:

  • Co-immunoprecipitation of HOXB3 with:

    • β-catenin

    • TCF/LEF factors

    • APC/destruction complex components

  • Chromatin conformation capture (3C/Hi-C) to identify:

    • Chromatin looping interactions

    • Co-localization with CTCF binding sites

• Transcriptional targets:

  • ChIP-seq for HOXB3 and H3K27ac

  • Identify super-enhancer regions regulated by WNT and HOXB3

  • RNA-seq following HOXB3 modulation with/without WNT activation

Research has shown that WNT3A and APC deficiency can lead to HOXB3 isolation from the destruction complex, nuclear translocation, and transcriptional regulation of multiple WNT pathway genes, suggesting a positive feedback loop in cancer progression .

What methods are effective for studying HOXB3's role in hematopoietic differentiation and lymphoid development?

HOXB3 overexpression perturbs normal hematopoietic differentiation, particularly affecting lymphoid development :

Experimental approaches:

• HOXB3 expression analysis during differentiation:

  • Flow cytometry with FITC-conjugated HOXB3 antibodies

  • Sorting of developmental subsets followed by qPCR/Western blot

  • Single-cell approaches to capture heterogeneity

  Cell Population	Markers	Expected HOXB3 Expression
  HSC	c-Kit+Flk-2-Lin-Sca-1+	High
  CMP	c-Kit+Lin-/loSca-1-CD34+CD16lo	Intermediate
  GMP	c-Kit+Lin-/loSca-1-CD34+CD16+	Low-Intermediate
  CLP	Lin-Sca-1loc-kitloCD127+	Low
  Mature cells	Lineage+	Very low/undetectable

• Functional assessment models:

  • Retroviral transduction of HOXB3 into bone marrow

  • Conditional knockout approaches

  • Transplantation studies to assess:

    • Thymic development

    • B-cell lymphopoiesis

    • Myeloid differentiation

• Molecular mechanisms:

  • RNA-seq to identify HOXB3-regulated genes

  • ChIP-seq to identify direct targets

  • Protein interaction partners through co-IP/mass spectrometry

Research indicates that HOXB3 overexpression results in:

• Reduced thymic size

• 24-fold decrease in CD4+CD8+ thymocytes

• 3-fold increase in CD4-CD8- thymocytes with high γδ TCR+ cells

• Absence of IL-7-responsive pre-B progenitors

• Elevated granulocyte-macrophage colony-forming cells

These findings suggest that HOXB3 regulates the balance between myeloid and lymphoid differentiation, potentially through controlling proliferation rates and lineage commitment decisions.