HOXB4 Antibody, FITC conjugated

What is HOXB4 and why is it significant in research?

HOXB4 is a homeodomain-containing transcription factor with diverse roles in embryonic development and the regulation of adult stem cells. It promotes proliferation of hematopoietic stem cells and can both activate and repress apoptosis . Research has shown that HOXB4 plays a critical role in hematopoietic stem cell (HSC) self-renewal and regeneration . Understanding HOXB4 expression and function has significant implications for stem cell research and potential therapeutic applications.

What applications is the HOXB4 Antibody, FITC Conjugated suitable for?

The HOXB4 Polyclonal Antibody, FITC Conjugated is suitable for multiple applications including:

• Western Blotting (WB)

• Flow Cytometry (FCM)

• Immunofluorescence on paraffin-embedded tissues (IF/IHC-P)

• Immunofluorescence on frozen tissues (IF/IHC-F)

• Immunocytochemistry (IF/ICC)

This versatility makes it valuable for researchers investigating HOXB4 expression across different experimental platforms.

What are the optimal dilutions for different applications of HOXB4 Antibody, FITC Conjugated?

These dilution ranges should be optimized based on specific experimental conditions and sample types .

What is the appropriate storage condition for HOXB4 Antibody, FITC Conjugated?

The antibody should be stored at -20°C. To maintain antibody integrity, it is recommended to aliquot into multiple vials to avoid repeated freeze-thaw cycles . The storage buffer typically contains 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol to help maintain stability during storage .

How should validation of HOXB4 expression be performed?

Western blotting is the gold standard for validating HOXB4 expression levels. When validating HOXB4 overexpression in experimental models (such as lentivirus-transfected cells), Western blotting should be used to confirm significant increases in HOXB4 protein expression compared to wild-type controls . Alternative validation methods include qRT-PCR for mRNA expression analysis and immunofluorescence staining to assess cellular localization.

What controls should be included when using HOXB4 Antibody, FITC Conjugated?

For robust experimental design, include:

• Positive control: Tissues or cell lines known to express HOXB4 (e.g., hematopoietic stem cells)

• Negative control: Tissues or cell lines with minimal HOXB4 expression

• Isotype control: Rabbit IgG, FITC conjugated with the same concentration as the primary antibody

• For overexpression studies: Empty vector controls (BMSC Vector) as demonstrated in validation protocols

• For knockout studies: Wild-type controls with normal HOXB4 expression

What is the optimal multiplicity of infection (MOI) for lentiviral HOXB4 overexpression studies?

When designing lentiviral HOXB4 overexpression experiments, test multiple MOI values (1, 10, 50, and 100) to determine the optimal condition. Select the MOI that achieves approximately 80% infection efficiency while maintaining good cell viability . Monitor fluorescence expression under a fluorescence microscope after 72 hours to assess transfection efficiency .

How can researchers distinguish between physiological and pathological HOXB4 expression levels?

This is a critical consideration as research has shown that while physiological HOXB4 levels promote normal hematopoietic stem cell function, high-level ectopic HOXB4 expression can have detrimental effects including impaired myeloerythroid differentiation and reduced B-cell output . Researchers should:

• Include appropriate controls for baseline expression

• Perform dose-response experiments to determine concentration thresholds

• Correlate HOXB4 expression levels with functional outcomes

• Consider using inducible expression systems to tightly control HOXB4 levels

How does HOXB4 deficiency affect hematopoietic development?

Studies with Hoxb4-deficient mice have revealed that:

• These mice exhibit significantly reduced cellularity in spleen and bone marrow

• There is a subtle reduction in red blood cell counts and hemoglobin values

• A mild reduction occurs in the numbers of primitive progenitors and stem cells in adult bone marrow and fetal liver

• Lineage distribution remains normal despite these deficiencies

• Cell cycle kinetics of primitive progenitors appears normal during endogenous hematopoiesis

• Defects in proliferative responses of bone marrow Lin-Sca1+c-kit+ stem and progenitor cells have been observed in culture and after transplantation

These findings suggest that while Hoxb4 is not required for HSC generation or steady-state hematopoiesis maintenance, it plays a role in regulating cellularity and proliferative capacity.

How does HOXB4 compare to its paralog HOXC4 in hematopoietic stem cell expansion?

HOXB4 and HOXC4 display important molecular analogies and have similar temporal and spatial expression patterns during embryogenesis. Research indicates that both molecules likely influence the expression of the same set of genes . Transcriptomic analysis reveals that CD34+ cells exposed to either HOXB4 or HOXC4 exhibit virtually identical gene expression profiles . Key hematopoietic factors and signaling pathway molecules affected include KLF10, HNRPDL, IKZF, and hypoxia, myc, IGF-1, 14-3-3, and angiopoietin-1 signaling pathways . HOXC4 has been investigated as an alternative to HOXB4 for expanding human hematopoietic cells, potentially offering improved expansion efficacy in human cells compared to HOXB4.

How can non-specific binding be minimized when using HOXB4 Antibody, FITC Conjugated?

To reduce non-specific binding in FITC-conjugated antibody applications:

• Optimize blocking conditions (use 1-5% BSA or normal serum from the same species as the secondary antibody)

• Ensure proper antibody dilution (start with manufacturer's recommendations and adjust as needed)

• Include appropriate washing steps between antibody incubations

• Use proper negative and isotype controls to distinguish specific from non-specific signals

• Pre-absorb the antibody if cross-reactivity is observed with unintended targets

What strategies can address poor fluorescent signal when using HOXB4 Antibody, FITC Conjugated?

If experiencing weak fluorescent signal:

• Verify the storage conditions of the antibody (improper storage can lead to fluorophore degradation)

• Check sample preparation protocols - ensure proper fixation and permeabilization

• Increase antibody concentration within recommended ranges

• Extend incubation time (overnight at 4°C may improve signal)

• Ensure protection from light during all stages to prevent photobleaching

• Consider signal amplification methods if endogenous HOXB4 levels are low

How is HOXB4 antibody being used in bone marrow stem cell protection studies?

Recent research has investigated the protective effects of HOXB4-overexpressing bone marrow mesenchymal stem cells (BMSCs) in various models. In co-culture experiments, HOXB4-overexpressing BMSCs demonstrated protective effects against lipopolysaccharide (LPS)-induced damage to endothelial cells . This suggests potential therapeutic applications for HOXB4 in protecting vascular integrity in inflammatory conditions. Researchers studying this application should consider co-culture experimental designs that include appropriate controls (wild-type BMSCs, vector-transfected BMSCs) and functional readouts to assess protective effects .

What are the considerations when studying HOXB4's role in genetic regulation?

Quantitative analysis of mRNA from fetal liver has revealed that Hoxb4 deficiency alone changes the expression levels of several other Hox genes and genes involved in cell cycle regulation . When investigating HOXB4's regulatory functions:

• Use comprehensive gene expression profiling approaches (RNA-seq or microarrays)

• Validate key findings using qRT-PCR

• Consider the interconnected nature of Hox gene regulation

• Include analyses of cell cycle-related genes

• Compare findings across different hematopoietic cell populations and developmental stages