HES7 Antibody, FITC conjugated

What is HES7 and why is it an important target for antibody-based research?

HES7 is a 225 amino acid transcriptional repressor protein localized to the nucleus that represses transcription of N box- and E box-containing promoters. It plays a crucial role in somite formation in the presomitic mesoderm and may be essential for coordinated somite segmentation by acting as a segmentation clock. HES7 contains one basic helix-loop-helix (bHLH) domain and one Orange domain .

The significance of HES7 as a research target stems from its involvement in developmental processes and disease associations. Mutations in HES7 have been found to cause spondylocostal dysostosis, an autosomal recessive disorder characterized by deformities of the chest and spine . HES7 plays a major role in segmentation, and its absence leads to segmentation defects. HES7 is expressed cyclically in the presomitic mesoderm over a 2-hour period, regulating the cyclic expression of the Fgf signaling inhibitor Dusp4 and linking Notch and Fgf oscillations in phase .

What are the fundamental characteristics of FITC conjugation in antibody applications?

FITC (Fluorescein isothiocyanate) is currently the most commonly used fluorescent dye for immunofluorescence and flow cytometry analysis. FITC is a small organic molecule that conjugates to proteins via primary amines (lysine residues). Typically, between 3 and 6 FITC molecules are conjugated to each antibody; higher conjugations can result in solubility problems and internal quenching, leading to reduced brightness .

Fluorescein is typically excited by the 488 nm line of an argon laser, and emission is collected at 530 nm . The conjugation process is influenced by several critical factors including reaction temperature, pH, and protein concentration. Optimal labeling is generally achieved at room temperature, pH 9.5, and an initial protein concentration of 25 mg/ml, with a reaction time of 30-60 minutes .

What are the recommended protocols for using FITC-conjugated HES7 antibodies in immunofluorescence studies?

For immunofluorescence applications using FITC-conjugated HES7 antibodies, the recommended dilution range is 1:50-200 for immunohistochemistry on paraffin-embedded tissues (IHC-P) . The following methodological approach is suggested:

• Sample preparation: Prepare formalin-fixed paraffin-embedded (FFPE) tissue sections, thawed frozen samples, or cell suspensions according to standard protocols.

• Antigen retrieval: Perform heat-induced epitope retrieval if working with FFPE sections.

• Blocking: Block non-specific binding with appropriate serum or protein blocking solution.

• Primary antibody incubation: Apply the FITC-conjugated HES7 antibody at the recommended dilution (1:50-200) and incubate according to established protocols (typically 1-2 hours at room temperature or overnight at 4°C).

• Washing: Wash thoroughly with PBS to remove unbound antibody.

• Counterstaining and mounting: Counterstain nuclei if desired and mount with an anti-fade mounting medium suitable for fluorescence microscopy.

• Visualization: Examine using a fluorescence microscope with appropriate filter sets for FITC (excitation at 488 nm, emission at 530 nm) .

When analyzing results, remember that HES7 is localized to the nucleus, so nuclear staining pattern should be expected in positive cells .

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

FITC-conjugated antibodies should be stored at -20°C for up to one year from the date of receipt . Critical storage recommendations include:

• Avoid repeated freeze-thaw cycles: Aliquot the antibody into smaller volumes before freezing to minimize freeze-thaw cycles, which can degrade both the antibody and the fluorophore.

• Protect from light: FITC is light-sensitive, so all FITC-conjugated antibodies should be protected from light during storage and handling to prevent photobleaching .

• Storage buffer considerations: FITC-conjugated antibodies are typically stored in a buffer containing stabilizers such as BSA (5 mg/mL) and 50% glycerol in PBS (pH 7.4) .

• Working solution stability: Once diluted for use, FITC-conjugated antibodies should ideally be used the same day. If necessary, diluted antibodies can be stored at 4°C for short periods (1-2 days) but should be protected from light.

• Monitor for precipitation: Before use, check for any visible precipitation. If present, centrifuge the antibody solution before use.

What are the optimal conditions for FITC conjugation to antibodies to ensure maximum functionality?

The optimization of FITC conjugation to antibodies requires careful control of several parameters to achieve maximum functionality:

For researchers performing their own conjugation, it's important to note that the reactive fluorescein molecule (FITC) is unstable once solubilized and should be used immediately . The separation of optimally labeled antibodies from under- and over-labeled proteins can be achieved by gradient DEAE Sephadex chromatography .

To determine the fluorescein/protein (F/P) ratio of the conjugate, measurements of absorbance at 280 nm (protein) and 495 nm (FITC) can be taken. The optimal F/P ratio typically ranges from 3 to 6, as higher conjugations can result in solubility problems and internal quenching .

What controls should be included when using FITC-conjugated HES7 antibodies in experimental systems?

When designing experiments with FITC-conjugated HES7 antibodies, the following controls should be included to ensure valid and interpretable results:

• Negative controls:

  • Isotype control: A FITC-conjugated rabbit IgG (for HES7 rabbit polyclonal antibody) with no specificity for the target

  • Secondary antibody-only control (if using indirect detection methods)

  • Unstained samples to assess autofluorescence

  • Samples from HES7 knockout models or HES7-negative tissues/cells

• Positive controls:

  • Tissues or cells known to express HES7, such as presomitic mesoderm tissues

  • Recombinant HES7 protein expressed in model systems

• Specificity controls:

  • Peptide competition assay: Pre-incubation of the antibody with excess HES7 protein/peptide should abolish specific staining

  • Parallel staining with a different anti-HES7 antibody (different clone or different detection method)

• Technical controls:

  • Single-color controls for compensation when performing multicolor flow cytometry

  • Fluorescence minus one (FMO) controls for setting gates in flow cytometry

  • Serial dilution of the antibody to determine optimal working concentration

These controls are essential for distinguishing specific signal from background and for troubleshooting any unexpected results.

How can researchers troubleshoot weak fluorescence signals when using FITC-conjugated HES7 antibodies?

When encountering weak fluorescence signals with FITC-conjugated HES7 antibodies, researchers should systematically troubleshoot using this methodological approach:

• Antibody-related factors:

  • Check antibody storage conditions (improper storage can lead to degradation)

  • Verify antibody concentration and increase if necessary (working within the recommended 1:50-200 range)

  • Consider antibody age (FITC has a finite shelf-life even when properly stored)

  • Evaluate F/P ratio if known (suboptimal conjugation can reduce signal intensity)

• Sample-related factors:

  • Ensure appropriate sample fixation and permeabilization

  • Verify target protein expression levels (HES7 may be expressed in a cyclical manner)

  • Optimize antigen retrieval methods for FFPE samples

  • Reduce autofluorescence using appropriate quenching methods

• Technical considerations:

  • Adjust microscope settings (exposure time, gain, etc.)

  • Ensure proper filter sets for FITC (excitation: 488 nm, emission: 530 nm)

  • Use an anti-fade mounting medium to prevent photobleaching

  • Consider signal amplification methods such as tyramide signal amplification

• Alternative approaches:

  • Try an indirect detection method with a primary anti-HES7 antibody and a secondary FITC-conjugated antibody

  • Consider using a brighter fluorophore-conjugated anti-HES7 antibody if available

  • Use a more sensitive detection system or instrument

How can FITC-conjugated HES7 antibodies be used to study spondylocostal dysostosis?

FITC-conjugated HES7 antibodies can be valuable tools for investigating spondylocostal dysostosis type 4 (SCDO4), a disorder caused by HES7 mutations. Research applications include:

• Protein localization studies: FITC-conjugated HES7 antibodies can be used to compare subcellular localization of wild-type and mutant HES7 proteins in patient-derived cells or transfected cell models.

• Expression pattern analysis: These antibodies can help visualize temporal and spatial expression patterns of HES7 in developmental models and patient tissues.

• Functional impact assessment: Researchers can investigate how HES7 mutations affect interactions with target DNA sequences (N-box and E-box containing promoters) through chromatin immunoprecipitation followed by visualization of binding sites .

• Phenotype-genotype correlation: By studying HES7 expression in tissues from patients with different HES7 variants (such as the c.226+1G>A splicing variant), researchers can better understand the molecular basis of phenotypic variation .

• Pathway interaction studies: FITC-conjugated HES7 antibodies can help visualize interactions between HES7 and components of the Notch and Fgf signaling pathways, which are critical for initiating and maintaining HES7 oscillation in the presomitic mesoderm .

When designing such studies, it's important to consider that mutations in HES7 may affect antibody binding, depending on the epitope recognized by the antibody. Therefore, epitope mapping and validation in the specific mutant context are recommended.

What methodological considerations are important when studying the cyclical expression of HES7 using FITC-conjugated antibodies?

HES7 is expressed cyclically in the presomitic mesoderm over a 2-hour period, which presents unique challenges for immunofluorescence studies . Key methodological considerations include:

• Temporal sampling: Due to the cyclic nature of HES7 expression, timed sample collection is critical. Researchers should establish a precise collection timeline, ideally spanning multiple cycles to capture the full expression dynamics.

• Spatial considerations: HES7 expression patterns vary spatially within the presomitic mesoderm. Using FITC-conjugated antibodies with confocal microscopy allows for detailed mapping of expression patterns along the anterior-posterior axis.

• Live imaging adaptations: For studying dynamic expression, consider using FITC-conjugated Fab fragments of HES7 antibodies, which can penetrate live tissues more effectively than full IgG molecules.

• Quantification methods: Develop robust quantification protocols to measure fluorescence intensity changes over time. This may include:

  • Image acquisition at fixed exposure settings

  • Background subtraction and normalization protocols

  • Region of interest (ROI) analysis corresponding to presomitic mesoderm segments

• Correlation with signaling pathways: Design co-localization experiments with markers of Notch and Fgf signaling pathways, as these regulate HES7 oscillation. HES7 oscillation is initiated by Fgf signaling and propagated/maintained by Notch signaling .

• Cross-validation: Complement protein-level studies with mRNA analysis (such as in situ hybridization) to distinguish between transcriptional and post-transcriptional regulation of the cyclical pattern.

• Fixation timing: Develop rapid fixation protocols to "freeze" the expression pattern at specific cycle points, minimizing artifacts due to continued oscillation during processing.

How can FITC-conjugated HES7 antibodies be optimized for flow cytometry applications?

While the Anti-HES7 Rabbit Polyclonal Antibody (FITC) is primarily recommended for immunofluorescence applications , researchers may adapt it for flow cytometry with these methodological considerations:

• Sample preparation protocols:

  • For detecting nuclear HES7, use permeabilization protocols optimized for nuclear antigens

  • Consider fixation with formaldehyde followed by permeabilization with methanol or commercial nuclear permeabilization buffers

  • Maintain cells in suspension throughout the staining procedure to ensure uniform antibody access

• Antibody titration:

  • Perform a titration series (typically starting from 1:25 to 1:200) to determine optimal signal-to-noise ratio

  • Plot staining index versus antibody concentration to identify the optimal dilution

• Compensation considerations:

  • Prepare single-color controls for proper compensation

  • When multiplexing with other fluorophores, consider spectral overlap with FITC (emission peak at 530 nm)

  • Use fluorophores with minimal spectral overlap for other targets in the panel

• Gating strategy development:

  • Use unstained and isotype controls to establish negative populations

  • Consider cell cycle status when analyzing nuclear transcription factors like HES7

  • Establish gates based on known positive and negative control cell populations

• Signal amplification options:

  • If direct FITC conjugation provides insufficient signal, consider biotin-streptavidin amplification systems

  • Evaluate the use of brighter fluorochromes through secondary antibody approaches if direct detection is suboptimal

• Analysis considerations:

  • Report data as percentage positive cells and median fluorescence intensity

  • Consider using biexponential display for proper visualization of populations

The typical excitation of FITC at 488 nm makes it compatible with standard flow cytometry laser configurations, though researchers should be aware of the potential for photobleaching during extended sample preparation or acquisition .

What are the considerations for using FITC-conjugated HES7 antibodies in combination with other fluorescent probes?

When designing multiplex immunofluorescence studies using FITC-conjugated HES7 antibodies alongside other fluorescent probes, researchers should consider:

• Spectral compatibility:

  • FITC has excitation maximum at 488 nm and emission maximum at 530 nm

  • Select companion fluorophores with minimal spectral overlap, such as:

    • DAPI for nuclear counterstaining (excitation ~350 nm, emission ~450 nm)

    • Texas Red or Cy3 for additional targets (emission >580 nm)

    • Far-red fluorophores like Cy5 or Alexa Fluor 647

• Sequential staining strategies:

  • For multiple primary antibodies from the same host species, consider sequential staining with complete blocking between steps

  • Use directly conjugated primary antibodies when possible to avoid cross-reactivity

• Microscopy setup optimization:

  • Configure microscope filter sets to minimize bleed-through

  • Acquire single-color controls to establish appropriate exposure settings

  • Consider spectral unmixing for fluorophores with partial overlap

• Co-localization analysis methodologies:

  • When studying interactions between HES7 and other proteins:

    • Use appropriate co-localization coefficients (Pearson's, Manders', etc.)

    • Implement proper thresholding to distinguish specific from background signal

    • Consider super-resolution microscopy for detailed co-localization studies

• Quantitative considerations:

  • Standardize image acquisition parameters across experimental groups

  • Use calibration standards if performing quantitative fluorescence measurements

  • Account for potential photobleaching differences between fluorophores

• Biological significance:

  • Design combinations that answer specific biological questions about HES7 function

  • Consider probing for interaction partners in the Notch and Fgf signaling pathways

  • Include markers for cellular compartments relevant to HES7 function (nuclear envelope, transcription factories)

When studying the cyclical expression pattern of HES7, carefully planned temporal sampling combined with multiplex staining can provide insights into the relationship between HES7 oscillation and other dynamic cellular processes.