MEIS3 Antibody, FITC conjugated

What is MEIS3 and why would researchers target it with antibodies?

MEIS3 is a homeobox protein functioning as a transcriptional regulator that directly modulates PDPK1 expression and promotes survival of pancreatic beta-cells. It also regulates expression of NDFIP1, BNIP3, and CCNG1 . MEIS3 belongs to the TALE-type class of homeobox gene family, with its protein showing high sequence similarity to MEIS1 and MEIS2 . MEIS1 was first described in a leukemia mouse model, suggesting potential roles for the MEIS family in hematopoietic processes . Researchers target MEIS3 to understand transcriptional regulation mechanisms in development and disease states, particularly in contexts where homeodomain proteins orchestrate cellular differentiation and tissue patterning.

FITC (fluorescein isothiocyanate) conjugation involves covalent attachment of the fluorophore to antibody molecules, typically via primary amine groups on lysine residues. The conjugation process must be carefully controlled to maintain antibody activity while providing sufficient fluorescence signal. As demonstrated with other antibody types, FITC conjugation preserves antibody activity while enabling direct visualization in applications like immunohistochemistry and flow cytometry .

Comparing FITC with enzymatic labels like peroxidase shows that while peroxidase conjugation (especially using glutaraldehyde methods) causes considerable loss of antibody and enzyme activity, FITC conjugation typically preserves antibody activity effectively . This preservation of functionality makes FITC an excellent choice for applications requiring both sensitivity and retention of binding specificity.

How should researchers optimize protocols for FITC-conjugated MEIS3 antibody in flow cytometry?

For optimal flow cytometry results with FITC-conjugated MEIS3 antibodies, researchers should:

• Determine optimal antibody concentration: Titrate the antibody to find the concentration that gives maximum signal-to-noise ratio. Starting dilutions between 1:100-1:200 are recommended based on similar antibody applications .

• Implement proper fixation and permeabilization: Since MEIS3 is a nuclear protein, thorough permeabilization is essential. A protocol similar to that used for detecting intracellular markers in human hematopoietic stem and progenitor cells (HSPCs) can be adapted, using PE-conjugated markers for surface antigens followed by fixation, permeabilization, and FITC-MEIS3 antibody staining .

• Set appropriate instrument settings: FITC excites at 494nm and emits at 519nm, requiring proper compensation when used in multicolor panels, especially with PE (which has spectral overlap).

• Include proper controls: Use isotype controls conjugated to FITC at the same protein concentration, and include both positive and negative controls for MEIS3 expression based on known expression patterns in cell lines like MCF7 and HepG2 .

What are the most effective validation methods to confirm MEIS3 antibody specificity?

Comprehensive validation of MEIS3 antibody specificity should employ multiple approaches:

• Western blot analysis: Confirm the antibody detects bands at expected molecular weights (approximately 41-46 kDa, depending on the isoform). MCF7 and HepG2 cell lysates have been used successfully to validate MEIS3 antibodies at dilutions of approximately 1:5000 .

• Knockout/knockdown controls: Use MEIS3 knockout or siRNA-mediated knockdown samples to confirm signal elimination.

• Peptide competition: Pre-incubate the antibody with the immunizing peptide (amino acids 1-261 of human MEIS3 for OTI2E12 clone) to confirm signal abolishment .

• Cross-reactivity testing: Test reactivity across multiple species where sequence conservation exists (human, mouse, rat) to confirm epitope recognition patterns .

• Immunoprecipitation followed by mass spectrometry: For definitive confirmation of antibody target specificity.

How can researchers address autofluorescence issues when using FITC-conjugated antibodies in tissue sections?

Autofluorescence presents a significant challenge when using FITC-conjugated antibodies, particularly in tissues containing lipofuscin, collagen, or elastin. Researchers can implement these strategies:

• Autofluorescence quenching: Pretreat sections with Sudan Black B (0.1-0.3% in 70% ethanol) for 10-20 minutes, followed by thorough washing.

• Alternative conjugates consideration: When autofluorescence persists in the green spectrum, consider switching to longer wavelength fluorophores or enzyme-based detection. Peroxidase conjugates prepared with glutaraldehyde can provide comparable sensitivity to FITC conjugates in immunohistochemical applications .

• Spectral unmixing: On confocal microscopes with spectral detectors, perform spectral unmixing to separate FITC signal from autofluorescence.

• Optimal fixation: Minimize aldehyde-induced autofluorescence by using shorter fixation times or alternative fixatives when possible.

What are the critical factors affecting antibody performance in MEIS3 detection systems?

Several factors can significantly impact MEIS3 antibody performance:

• Epitope accessibility: The MEIS3 protein contains distinct functional domains including the homeodomain. Antibodies targeting different regions (such as amino acids 1-110 versus 1-261) may perform differently depending on protein conformation and interactions .

• Sample preparation method: Different fixation and permeabilization protocols substantially affect nuclear antigen detection. Paraformaldehyde fixation followed by Triton X-100 permeabilization typically works well for nuclear transcription factors.

• Antibody concentration and incubation conditions: For optimal results in Western blot applications, MEIS3 antibodies have been successfully used at dilutions of 1:2000-1:5000 , while immunohistochemistry typically requires more concentrated preparations (approximately 1:150) .

• Storage conditions: Antibody performance can degrade with improper storage or repeated freeze-thaw cycles. Storage at 4°C (for conjugated antibodies) or -20°C (for unconjugated formats) is typically recommended .

How can FITC-conjugated MEIS3 antibodies be integrated into multiplex imaging systems?

Multiplex imaging with FITC-conjugated MEIS3 antibodies requires strategic planning:

• Sequential staining protocols: To minimize cross-reactivity in multiplex panels, consider sequential rather than cocktail staining approaches, especially when combining antibodies from the same host species.

• Complementary fluorophore selection: When designing panels, pair FITC (excitation: 494nm, emission: 519nm) with fluorophores having minimal spectral overlap, such as APC (excitation: 650nm, emission: 660nm) used for CD133 detection in HSPCs studies .

• Signal amplification strategies: For low-abundance targets, consider using biotin-conjugated primary antibodies (like OTI2E12) followed by streptavidin-fluorophore secondaries to enhance signal while maintaining multiplexing capability .

• Cyclic immunofluorescence: For highly complex panels, implement cyclic immunofluorescence with fluorophore inactivation between rounds to exceed conventional fluorophore limits.

What approaches enable quantitative analysis of MEIS3 expression in cellular subpopulations?

Quantitative analysis of MEIS3 expression requires rigorous methodological approaches:

• Standardized flow cytometry: Implement quantitative flow cytometry using calibration beads with known quantities of fluorophore to convert fluorescence intensity to molecules of equivalent soluble fluorophore (MESF) values.

• Digital pathology integration: Combine MEIS3 immunofluorescence with whole slide imaging and machine learning-based image analysis to quantify expression levels across tissue sections.

• Single-cell analysis pipelines: Integrate FITC-MEIS3 antibody staining with single-cell RNA sequencing workflows to correlate protein expression with transcriptomic profiles.

• Mass cytometry adaptation: Consider using metal-conjugated MEIS3 antibodies (such as the CyTOF-ready OTI3E12 clone) for highly multiplexed, quantitative analysis of MEIS3 in complex cellular hierarchies .

How do FITC-conjugated antibodies compare with enzyme-conjugated alternatives for MEIS3 detection?

How are MEIS3 antibodies contributing to hematopoietic stem cell research?

MEIS3 antibodies are becoming valuable tools in hematopoietic stem cell (HSC) research due to the important roles of MEIS family proteins in hematopoiesis:

• HSC self-renewal mechanisms: MEIS1, a close homolog of MEIS3, has been identified as a key regulator of HSC self-renewal, suggesting potential parallel functions for MEIS3 that researchers are now investigating using specific antibodies .

• Development of small molecule inhibitors: Researchers have established screening strategies for MEIS inhibitors that could modulate HSC activity, with antibody-based assays serving as critical validation tools for target engagement .

• Flow cytometry characterization: MEIS3 antibodies can be incorporated into flow cytometry panels alongside established HSC markers like CD34 and CD133 to investigate correlations between MEIS family expression and stem cell functionality .

• Human-mouse comparative studies: Given the reactivity of several MEIS3 antibody clones across human, mouse, and rat samples, researchers can perform comparative studies of MEIS3 function across species models of hematopoiesis .

What technical innovations are improving the specificity and sensitivity of fluorophore-conjugated antibodies for transcription factor detection?

Recent technical advances enhancing fluorophore-conjugated antibody performance include:

• Site-specific conjugation methods: Unlike traditional random conjugation to lysines, site-specific attachment of FITC to engineered cysteine residues or enzymatically modified sites preserves antibody binding regions, improving both sensitivity and batch consistency.

• Conjugation-ready formats: Antibody formats specifically designed for fluorochrome, metal isotope, oligonucleotide, and enzyme labeling improve conjugate performance across multiple detection platforms .

• Enhanced fluorophores: Next-generation fluorescent dyes with improved brightness, photostability, and narrower emission spectra are replacing traditional FITC for superior signal-to-noise ratios and reduced bleed-through in multiplex applications.

• Proximity-based signal amplification: Techniques combining primary antibody detection with rolling circle amplification or proximity ligation provide exponential signal enhancement for low-abundance nuclear factors like MEIS3.