LGALS4 Antibody, FITC conjugated

What is Galectin-4 and why is it an important research target?

Galectin-4 (LGALS4) is a tandem-repeat galectin consisting of two carbohydrate recognition domains (CRDs) connected by a flexible linker. It is naturally expressed in epithelial cells throughout the gastrointestinal tract, including the antrum, ileum, colon, and rectum . The protein has gained significant research interest due to its dual role in modulating immunity and its involvement in cancer progression. Galectin-4 can bind to glycosylated amino acid residues, particularly N-glycosylation residues on T cell surface proteins such as CD3ε/δ, potentially inducing T cell apoptosis . This immunomodulatory function makes Galectin-4 particularly relevant in cancer research, as elevated levels have been associated with tumor progression. The protein has demonstrated capabilities to exacerbate intestinal inflammation through increased IL6 production in certain contexts, highlighting its complex role in immune regulation .

How should I determine the optimal dilution for immunofluorescence applications using FITC-conjugated LGALS4 antibodies?

Determining the optimal dilution for FITC-conjugated LGALS4 antibodies in immunofluorescence applications requires systematic titration. Based on manufacturer recommendations for similar antibodies, starting dilutions typically range from 1:200 to 1:800 for immunofluorescence applications . To establish the optimal concentration for your specific experimental conditions, prepare a dilution series (e.g., 1:100, 1:200, 1:400, 1:800, 1:1600) and test these on positive control samples known to express Galectin-4, such as HT-29 cells which have been validated for LGALS4 detection . The optimal dilution will provide the best signal-to-noise ratio - strong specific staining with minimal background. For each dilution, include appropriate negative controls such as isotype controls and samples known to lack Galectin-4 expression (e.g., HCT-116 cells have been reported to lack surface-bound Galectin-4) . Document results systematically using standardized exposure settings across all samples, and quantify signal intensity and background levels. Remember that optimal dilutions may differ between applications (IF vs. flow cytometry) and across different tissue types due to variations in target abundance and accessibility.

What fixation and permeabilization protocols are recommended for detecting intracellular versus surface-bound Galectin-4?

The appropriate fixation and permeabilization protocol depends critically on whether you are targeting surface-bound or intracellular Galectin-4. For surface-bound Galectin-4 detection, as demonstrated in studies with LS-180 cells, a gentle fixation without permeabilization is essential . A recommended protocol includes:

• Fixing cells with 2-4% paraformaldehyde in PBS for 10-15 minutes at room temperature

• Washing three times with PBS

• Blocking with 1-5% BSA in PBS for 30-60 minutes

• Incubating with FITC-conjugated LGALS4 antibody at the optimal dilution (typically 1:200-1:800)

• Washing thoroughly with PBS before mounting and imaging

For intracellular Galectin-4 detection, permeabilization is required after fixation:

• Fix cells as described above

• Permeabilize with 0.1-0.5% Triton X-100 in PBS for 5-10 minutes

• Wash three times with PBS

• Follow the blocking and antibody incubation steps as above

The choice between these protocols significantly impacts results, as demonstrated in immunofluorescence studies where gal-4 was detectable at the cell surface in LS-180 cells but not in HCT-116 cells when using non-permeabilization conditions . When comparing surface-bound versus intracellular expression patterns, it is advisable to process parallel samples with both protocols to obtain a comprehensive understanding of Galectin-4 distribution.

How can I validate the specificity of my FITC-conjugated LGALS4 antibody?

Validating antibody specificity is critical for obtaining reliable research results. For FITC-conjugated LGALS4 antibodies, implement a multi-faceted validation approach:

• Positive and negative control cell lines: Use known Galectin-4 expressing cell lines like LS-180 and HT-29 as positive controls, and non-expressing lines like HCT-116 as negative controls .

• Blocking experiments: Pre-incubate the antibody with recombinant Galectin-4 protein before application to samples. Specific binding should be significantly reduced or eliminated.

• siRNA knockdown: Compare staining between wild-type cells and those treated with LGALS4-specific siRNA. Specific antibodies will show reduced signal in knockdown cells.

• Western blot correlation: Confirm that immunofluorescence results correlate with Western blot detection, verifying the expected molecular weight of 34-36 kDa for Galectin-4 .

• Multi-antibody validation: Compare staining patterns with alternative antibodies targeting different epitopes of Galectin-4.

Documentation should include images of all validation experiments with standardized acquisition settings, quantification of signal differences between positive and negative controls, and comparison of results across different validation methods. This comprehensive approach ensures confidence in subsequent experimental findings and facilitates troubleshooting if unexpected results occur.

How can I optimize protocols for detecting Galectin-4 in cancer tissue microarrays using FITC-conjugated antibodies?

Optimizing protocols for cancer tissue microarray (TMA) analysis with FITC-conjugated LGALS4 antibodies requires addressing several tissue-specific challenges. Begin with antigen retrieval optimization, as different cancer tissues may require specific conditions. Based on validated protocols, both citrate buffer (pH 6.0) and TE buffer (pH 9.0) have been successfully used for LGALS4 antibody staining, with TE buffer being the primary recommendation . For pancreatic tumors, where Galectin-4 has been identified as abundant and involved in immune evasion mechanisms, more stringent antigen retrieval may be necessary .

Create a testing matrix varying:

• Antigen retrieval buffers and durations

• Antibody concentrations (starting range: 1:50-1:1000 as recommended for IHC applications)

• Incubation times and temperatures

• Signal amplification systems if needed for low-expressing samples

For cancer TMAs specifically, always include:

• Serial sections stained with H&E for morphological correlation

• Normal tissue controls from matched organs

• Progressive cancer stages to assess expression changes

To address tissue autofluorescence, which is particularly problematic with FITC:

• Incorporate a Sudan Black B treatment (0.1-0.3% in 70% ethanol for 10-20 minutes)

• Use spectral unmixing during image acquisition if available

• Consider automated background subtraction algorithms during analysis

Document antibody performance across different cancer types, noting that Galectin-4 expression has been specifically validated in colon cancer and stomach cancer tissues . Maintain standardized image acquisition settings across all samples to enable valid quantitative comparisons.

What strategies can address non-specific binding when using FITC-conjugated LGALS4 antibody in flow cytometry?

Non-specific binding in flow cytometry with FITC-conjugated LGALS4 antibodies can significantly impact data interpretation. Studies detecting surface-bound Galectin-4 on cell lines like LS-180 and HT-29 demonstrated that proper controls are essential for distinguishing specific from non-specific signals . Implement these strategies to optimize specificity:

• Optimize blocking protocols:

  • Extended blocking (60+ minutes) with 5-10% normal serum from the same species as secondary antibody

  • Addition of 0.1-0.5% BSA and 0.05-0.1% Tween-20 to all wash buffers

  • Pre-incubation with unconjugated Fc receptor blocking antibodies for samples containing immune cells

• Refine antibody concentration:

  • Perform detailed titration experiments (typically starting at 1:100-1:500)

  • Plot signal-to-noise ratio against antibody concentration to identify optimal dilution

  • Consider that flow cytometry may require different optimal concentrations than microscopy

• Implement robust controls:

  • Isotype control antibodies conjugated to FITC at identical concentrations

  • Fluorescence minus one (FMO) controls

  • Known positive (LS-180) and negative (HCT-116) cell lines

  • Pre-adsorption controls with recombinant Galectin-4 protein

• Adjust acquisition settings:

  • Set voltage based on unstained controls

  • Implement compensation if using multiple fluorochromes

  • Use biexponential scaling for better visualization of negative populations

When analyzing the data, examine fluorescence histogram overlays of test samples versus controls to clearly distinguish specific binding. Calculate the signal-to-noise ratio as median fluorescence intensity of positive sample divided by isotype control. For surface-bound Galectin-4 analysis, results should show distinct peaks with increased fluorescence intensity compared to controls, similar to the pattern observed with LS-180 and HT-29 cells in published reports .

How does Galectin-4's role in immune evasion inform experimental design using FITC-conjugated antibodies?

Galectin-4's newly discovered role in immune evasion mechanisms should inform experimental design when using FITC-conjugated LGALS4 antibodies. Research has demonstrated that Galectin-4 induces apoptosis in T cells by binding N-glycosylation residues on CD3ε/δ, contributing to immunosuppression in pancreatic ductal adenocarcinoma (PDAC) . This finding suggests several important considerations for experimental design:

• Co-localization studies: Design dual-labeling experiments to investigate Galectin-4 interactions with:

  • T cell receptors (especially CD3 complex components)

  • N-glycosylation patterns on immune cells

  • Apoptotic markers to confirm functional outcomes

• Time-course experiments: Monitor Galectin-4 expression and localization during:

  • Tumor-immune cell interactions

  • Progression of tumor development

  • Response to immunotherapy treatments

• Functional correlation: Integrate FITC-LGALS4 antibody staining with:

  • Flow cytometric assessment of T cell apoptosis

  • Analysis of tumor-infiltrating lymphocyte populations

  • Measurement of inflammatory cytokine profiles

When designing these experiments, researchers should consider that reduced Galectin-4 expression has been associated with higher proportions of M1 macrophages, T cells, and antigen-presenting dendritic cells in tumors . This suggests examining multiple immune cell populations simultaneously when investigating Galectin-4's role. The experimental design should also account for microenvironmental factors, as studies have shown that the myeloid compartment and cancer-associated fibroblast subtypes are altered in tumors with different Galectin-4 expression levels . These considerations ensure that FITC-conjugated LGALS4 antibody applications yield meaningful insights into the protein's immunomodulatory functions beyond simple expression analysis.

What are the considerations for using FITC-conjugated LGALS4 antibodies in conjunction with therapeutic targeting strategies?

When utilizing FITC-conjugated LGALS4 antibodies in research related to therapeutic targeting of Galectin-4, several important considerations must be addressed to ensure scientifically sound experimental design and interpretation:

• Epitope accessibility in therapeutic contexts:

  • Determine whether the epitope recognized by your FITC-conjugated antibody overlaps with potential therapeutic binding sites

  • Assess whether binding of therapeutic agents alters detection by the FITC-conjugated antibody

  • Consider competitive binding assays between therapeutic candidates and labeled antibodies

• Monitoring therapeutic response:

  • Design time-course experiments to track Galectin-4 expression changes during treatment

  • Establish baseline expression levels across different tumor models

  • Develop quantitative imaging or flow cytometry protocols for consistent measurement

• Context-specific expression patterns:

  • Account for the dual role of Galectin-4 in different contexts (pro-inflammatory in some settings, immunosuppressive in others)

  • Consider that Galectin-4's effects depend on whether it is intracellular, surface-bound, or extracellular

  • Evaluate expression in both cancer cells and stromal/immune components

• Functional correlation with therapeutic outcomes:

  • Correlate Galectin-4 levels with T cell apoptosis rates

  • Monitor changes in tumor-infiltrating immune cell populations

  • Assess alterations in cancer-associated fibroblast subtypes before and after treatment

The identification of Galectin-4 as a promising drug target for overcoming immunosuppression in pancreatic ductal adenocarcinoma makes these considerations particularly relevant. Researchers should design experiments that not only detect Galectin-4 expression but also integrate functional readouts relevant to the therapeutic mechanism, such as restoration of T cell function or reversal of the immunosuppressive tumor microenvironment.

How do I design multi-parameter flow cytometry panels incorporating FITC-conjugated LGALS4 antibody?

Designing effective multi-parameter flow cytometry panels with FITC-conjugated LGALS4 antibody requires careful consideration of spectral overlap, fluorophore brightness, and antigen density relationships. Since FITC emits in the green spectrum (peak ~520nm), plan your panel to minimize spillover with other green-yellow fluorophores such as PE and AF532.

When incorporating FITC-conjugated LGALS4 antibody:

• Spectral considerations:

  • Reserve red and far-red channels (APC, AF647) for low-abundance antigens

  • Pair FITC with fluorophores having minimal spectral overlap like APC, AF647, or BV421

  • If using PE (yellow), ensure proper compensation controls are included

• Panel design strategy:

  • Based on studies showing Galectin-4's interaction with T cells , consider these marker combinations:

    Target	Suggested Fluorophore	Rationale
    LGALS4	FITC	Primary target protein
    CD3	APC or BV421	Interaction partner for Galectin-4
    Apoptosis markers (Annexin V)	PE-Cy7	Functional outcome of Galectin-4 binding
    CD4/CD8	AF647/BV510	T cell subset identification
    Activation markers	BV605/BV650	Functional status assessment

• Titration and optimization:

  • Determine optimal concentration of FITC-LGALS4 antibody through systematic titration

  • Test staining on known positive cell lines like LS-180 and HT-29

  • Include fluorescence minus one (FMO) controls for each channel

  • Prepare single-stained compensation controls for each fluorophore

• Analytical considerations:

  • Use appropriate gating strategies to identify Galectin-4 positive populations

  • Consider biexponential display for better visualization of populations

  • Include appropriate controls to distinguish surface-bound from intracellular Galectin-4

For studies investigating Galectin-4's role in immune evasion, design panels that allow simultaneous evaluation of Galectin-4 expression and immune cell function. This approach enables direct correlation between Galectin-4 levels and immunosuppressive effects within the same sample.

What confocal microscopy settings are optimal for visualizing FITC-conjugated LGALS4 antibody staining patterns?

Optimizing confocal microscopy settings for FITC-conjugated LGALS4 antibody requires balancing signal detection, resolution, and photobleaching considerations. Based on successful immunofluorescence visualization of surface-bound Galectin-4 in cell lines like LS-180 , the following settings are recommended:

• Excitation and emission parameters:

  • Excitation: 488 nm laser line (optimal for FITC)

  • Emission collection: 500-550 nm bandpass filter

  • Dichroic mirror: 495 nm longpass

• Image acquisition settings:

  • Pixel size: 0.1-0.2 μm for subcellular localization studies

  • Optical section thickness: 0.5-1.0 μm (adjust based on cell/tissue thickness)

  • Frame averaging: 2-4 frames to improve signal-to-noise ratio

  • Scan speed: Medium to slow for better signal quality

  • Line averaging: 2-4 lines to reduce noise

• Laser power and photobleaching management:

  • Start with low laser power (5-15%) to minimize photobleaching

  • Use bi-directional scanning to reduce exposure time

  • Implement ROI scanning for detailed analysis of specific cellular regions

  • Consider using antifade mounting media specifically optimized for FITC

• Detector optimization:

  • PMT voltage: Adjust to place the histogram peak in the middle range

  • Gain: Set to avoid saturation while maximizing dynamic range

  • Offset: Adjust to ensure background is slightly above zero

For differential analysis of surface versus intracellular Galectin-4, Z-stack acquisition is essential. Collect optical sections at 0.3-0.5 μm intervals through the entire cell volume (typically 10-20 sections for cultured cells). This approach allows three-dimensional reconstruction and precise localization of Galectin-4, which is critical when investigating its dual role as both an intracellular and surface-bound/secreted protein . For colocalization studies examining Galectin-4 interaction with immune cell receptors, use sequential scanning to eliminate crosstalk between channels.

How can I quantitatively analyze LGALS4 expression levels in different cellular compartments?

Quantitative analysis of Galectin-4 expression across different cellular compartments requires systematic image processing and analytical approaches. Given the evidence that Galectin-4 can be found intracellularly, surface-bound, and in the extracellular environment , compartment-specific quantification provides valuable insights into its biological functions.

Recommended quantitative workflow:

• Image acquisition standardization:

  • Maintain identical microscope settings across all samples

  • Include calibration standards in each imaging session

  • Collect z-stacks to capture the full cellular volume

• Image processing sequence:

  • Apply flat-field correction to address illumination non-uniformity

  • Implement background subtraction using rolling ball algorithm

  • Use deconvolution to improve signal localization (especially for thick samples)

• Compartment segmentation approaches:

  Compartment	Segmentation Method	Additional Markers
  Cell surface	Membrane detection algorithms	Membrane dyes (DiI, WGA)
  Cytoplasm	Threshold-based after nucleus exclusion	Cytoplasmic markers
  Nucleus	DAPI/Hoechst-based thresholding	-
  Extracellular	Outside cell boundary analysis	-

• Quantification metrics:

  • Mean fluorescence intensity per compartment

  • Integrated density (area × mean intensity)

  • Ratio of surface to intracellular signal

  • Colocalization coefficients with compartment markers (Pearson's, Manders')

• Statistical analysis:

  • Compare expression levels across different cell types (e.g., LS-180 vs. HT-29)

  • Normalize to total cellular expression

  • Calculate expression ratios between compartments

For extracellular Galectin-4 quantification, complement imaging with biochemical approaches such as immunoprecipitation from conditioned media, which has successfully detected secreted Galectin-4 from cell lines like HT-29 and LS-180 . When analyzing surface-bound Galectin-4, implement careful z-analysis with surface rendering to distinguish true membrane localization from near-membrane cytoplasmic signal. This multi-faceted quantitative approach enables correlation between Galectin-4 subcellular distribution and its diverse functions in normal physiology and disease states.

How can FITC-conjugated LGALS4 antibodies be used to investigate Galectin-4's role in cancer immunosuppression?

FITC-conjugated LGALS4 antibodies provide powerful tools for investigating Galectin-4's emerging role in cancer immunosuppression. Research has identified that extracellular Galectin-4 induces apoptosis in T cells by binding N-glycosylation residues on CD3ε/δ, contributing to immune evasion in pancreatic tumors . FITC-conjugated antibodies enable direct visualization of these interactions through several experimental approaches:

• Tumor microenvironment analysis:

  • Use multicolor immunofluorescence to simultaneously visualize:

    • Galectin-4 expression (FITC-conjugated anti-LGALS4)

    • T cell infiltration (anti-CD3)

    • Apoptotic markers (Annexin V, cleaved caspase-3)

  • Quantify spatial relationships between Galectin-4-expressing cancer cells and T cell populations

  • Create expression maps correlating Galectin-4 levels with immune cell density and apoptosis rates

• Mechanistic investigation of T cell apoptosis:

  • Implement time-lapse imaging using FITC-conjugated LGALS4 antibodies to track:

    • Initial Galectin-4 binding to T cell surface

    • Subsequent apoptotic events

    • Temporal relationship between binding and cell death

  • Correlate with flow cytometric analysis of apoptosis markers in the same samples

• Therapeutic intervention monitoring:

  • Assess changes in Galectin-4 expression following immunotherapy

  • Track redistribution of Galectin-4 between cellular compartments during treatment

  • Correlate with functional recovery of T cell populations

The experimental design should account for the finding that reduced Galectin-4 expression is associated with higher proportions of M1 macrophages, T cells, and antigen-presenting dendritic cells in tumors . This suggests including these additional immune cell markers in multiplex analysis. By implementing these approaches with FITC-conjugated LGALS4 antibodies, researchers can gain critical insights into the mechanisms of cancer immunosuppression and identify potential intervention points for therapeutic development.

What protocol modifications are needed when using FITC-conjugated LGALS4 antibodies in different cancer models?

Different cancer models require specific protocol modifications when using FITC-conjugated LGALS4 antibodies due to variations in Galectin-4 expression, tissue architecture, and microenvironmental factors. Based on the research literature and technical specifications, the following cancer-specific adaptations are recommended:

Colorectal Cancer Models:

• Optimal antibody dilution: 1:50-1:200 for IHC applications based on validated staining in human colon cancer tissue

• Antigen retrieval: TE buffer (pH 9.0) as the primary recommendation, with citrate buffer (pH 6.0) as an alternative

• Controls: Include normal colonic epithelium, which naturally expresses Galectin-4 , as a positive control

• Cell lines: LS-180 and HT-29 have been validated for surface-bound Galectin-4 detection

Pancreatic Cancer Models:

• Account for the abundant Galectin-4 expression reported in pancreatic tumors

• Implement dual staining with immune cell markers to visualize immunosuppressive mechanisms

• Include stroma analysis, as altered cancer-associated fibroblast subtypes correlate with Galectin-4 expression

• Consider comparison between wild-type and Galectin-4-knockout models to assess immune infiltration differences

Tissue-Specific Background Reduction:

• For highly autofluorescent tissues (liver, pancreas):

  • Increase blocking time (60+ minutes)

  • Add 0.1-0.3% Sudan Black B treatment after antibody incubation

  • Consider shorter wavelength fluorophores if FITC background is problematic

Sample Type Variations:

When implementing these modifications, it's essential to systematically document and compare staining patterns across different cancer models. This comparative approach will provide valuable insights into how Galectin-4 expression and localization vary across cancer types and may correlate with disease-specific immune evasion mechanisms or treatment responses.