ITGAX Antibody

What is ITGAX/CD11c and what is its significance in immunological research?

ITGAX (Integrin Subunit Alpha X), also known as CD11c, is a 145 kDa transmembrane glycoprotein that belongs to the integrin alpha chain family. It functions as part of the leukocyte adhesion molecule family, sharing the same beta subunit with CD11a (LFA-1), CD11b (MAC-1), and CD11d (ITGAD) but possessing a unique alpha chain . CD11c plays critical roles in phagocytosis, cell migration, and cytokine production by monocytes and macrophages. Additionally, it contributes to T-cell proliferation induction by Langerhans cells .

The significance of CD11c in immunological research stems from its selective expression on dendritic cells, making it a valuable marker for identifying and isolating these professional antigen-presenting cells. Research involving CD11c antibodies has been instrumental in advancing our understanding of innate immunity, antigen presentation, and inflammatory responses in various disease models and tissue microenvironments.

What are the primary applications for ITGAX antibodies in research settings?

ITGAX antibodies are utilized across multiple research applications with varying protocols and optimization requirements. The primary validated applications include:

When designing experiments, researchers should consider that CD11c expression is prominently observed on plasma membranes of monocytes, particularly in tissues such as human spleen, tonsillitis samples, lung cancer tissue, breast cancer tissue, liver cancer tissue, and appendicitis tissue . The selection of appropriate antibody clone and detection method should be guided by the specific cell populations and tissue contexts under investigation.

How should researchers determine the optimal antibody dilution for their specific experimental system?

Determining the optimal antibody dilution requires systematic titration to balance signal strength and specificity. While manufacturers provide recommended dilution ranges (such as 1:500-1:2000 for Western blot applications) , these should be considered starting points rather than definitive values. The optimization process should follow these methodological steps:

First, conduct a preliminary experiment using a broad dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000) with your specific sample type. Evaluate both signal intensity and background levels across this range. Second, narrow down to a more refined dilution series around the best-performing concentration. For IHC applications with ITGAX antibodies, significantly higher dilutions (1:10000-1:40000) may be optimal due to the high expression levels in certain tissues .

Critical variables that influence optimal dilution include sample type (cell line vs. primary tissue), fixation method, antigen abundance, and detection system sensitivity. For instance, enhanced chemiluminescence (ECL) detection systems for Western blots may allow for higher antibody dilutions compared to chromogenic detection methods. Researchers should document that "sample-dependent" outcomes are common with ITGAX antibodies, and validation data galleries should be consulted when available .

What strategies can researchers employ to validate ITGAX antibody specificity in their experimental system?

Validating antibody specificity is crucial for generating reliable data with ITGAX antibodies. A comprehensive validation strategy should incorporate multiple complementary approaches:

First, employ genetic controls whenever possible, such as ITGAX knockout or knockdown samples, which provide the most definitive test of antibody specificity. Second, perform peptide competition assays using the immunizing peptide (such as the CD11c/Integrin Alpha X fusion protein Ag11350) to demonstrate signal reduction when the antibody is pre-incubated with its target epitope.

Third, cross-validate using multiple antibody clones targeting different ITGAX epitopes, as convergent results strengthen confidence in specificity. For instance, comparing results between the 8E3 clone and the ITGAX/1284 clone can be informative . Fourth, correlate protein detection with mRNA expression data from RT-PCR or RNA-seq, as concordance between protein and transcript levels supports antibody specificity.

Finally, apply tissue and cell-type controls by testing the antibody on samples with known expression patterns. ITGAX antibodies should show strong reactivity with myeloid lineage cells like THP-1, HL-60, and U-937 cell lines , while demonstrating minimal background on ITGAX-negative cell types. These multi-faceted validation approaches ensure that experimental findings are genuinely reflecting ITGAX biology rather than artifacts.

How can researchers troubleshoot inconsistent ITGAX antibody staining patterns between different detection methods?

Inconsistencies in ITGAX staining patterns between methods like IHC, IF, and flow cytometry often stem from technical variables rather than biological differences. A systematic troubleshooting approach should consider several key factors:

First, evaluate fixation and epitope accessibility differences. ITGAX epitopes may be differentially preserved or exposed depending on fixation method. For IHC applications, recommended antigen retrieval with TE buffer pH 9.0 may be critical, though citrate buffer pH 6.0 provides an alternative approach for some tissue types . Formalin fixation can mask certain epitopes while preserving others, leading to method-specific detection patterns.

Second, consider the impact of antibody format and conjugation. Native unconjugated antibodies used in WB or IHC may perform differently from conjugated versions (Biotin, Cy3, Dylight488) used in fluorescence-based applications . Direct comparison of conjugated and unconjugated formats of the same clone can help isolate this variable.

Third, assess buffer composition and blocking reagents across protocols. Certain detergents or blocking proteins may affect epitope accessibility or create background with specific detection systems. Standardizing buffer components or systematically testing alternatives can identify problematic reagents.

Finally, implement multi-color analyses to distinguish true ITGAX signal from autofluorescence or non-specific binding. Co-staining with markers of ITGAX-positive cells (myeloid lineage markers) versus ITGAX-negative populations provides internal controls within the same sample to validate staining patterns across methods.

What are the considerations for cross-species reactivity when using ITGAX antibodies?

Cross-species reactivity varies considerably among ITGAX antibody clones and requires careful validation before application to non-human samples. When selecting antibodies for cross-species applications, researchers should consider:

First, sequence homology analysis between the target epitope regions across species provides a theoretical basis for cross-reactivity. While some ITGAX antibodies like the 8E3 clone demonstrate reactivity with human, mouse, and rat samples , others may be more species-restricted, such as those primarily validated on human samples (60258-1-Ig) .

Second, empirical validation is essential regardless of manufacturer claims. Positive and negative control samples from each target species should be tested alongside experimental samples. For instance, if an antibody claims mouse reactivity, mouse spleen tissue (rich in CD11c-positive cells) provides an appropriate positive control.

Third, optimization of experimental conditions for each species is necessary. Different species may require distinct antigen retrieval methods, antibody concentrations, or detection systems. Protocol modifications should be systematically evaluated and documented.

Lastly, researchers should consider species-specific background problems. Some secondary antibodies may react with endogenous immunoglobulins in certain species, necessitating blocking steps or alternative detection strategies. For example, when using mouse monoclonal antibodies on mouse tissues, specialized blocking systems or directly conjugated primary antibodies may be required to avoid background.

What are the optimal protocols for using ITGAX antibodies in multiplex immunofluorescence studies?

Multiplex immunofluorescence with ITGAX antibodies requires careful planning to maximize signal specificity while minimizing spectral overlap and antibody cross-reactivity. The following methodological approach is recommended:

First, select compatible antibody pairs based on host species, isotype, and available fluorophore conjugates. For ITGAX, mouse monoclonal antibodies like clone 8E3 (IgG isotype) or ITGAX/1284 can be paired with antibodies raised in different host species (rabbit, rat, etc.) to facilitate simultaneous detection. When multiple mouse monoclonals must be used, sequential staining with direct conjugates or isotype-specific secondaries becomes essential.

Second, optimize the staining sequence for tyramide signal amplification (TSA) or other multiplexing systems. ITGAX should typically be placed earlier in the staining sequence when using sequential TSA approaches, as its membrane localization pattern is less prone to interference from preceding rounds of staining compared to nuclear or cytoplasmic markers.

Third, implement robust controls for each marker in the panel. Single-stained controls, fluorescence-minus-one (FMO) controls, and isotype controls help identify and correct for spectral overlap and non-specific binding. Given the variable expression of ITGAX across different dendritic cell and macrophage populations, biological reference samples with known expression patterns provide valuable benchmarks.

Fourth, select compatible fluorophores based on tissue autofluorescence characteristics. In tissues with high autofluorescence (like lung or liver), longer-wavelength fluorophores (far-red) may provide better signal-to-noise for ITGAX detection compared to those in the green or yellow spectrum.

How can researchers integrate ITGAX antibody staining with tissue clearing techniques for 3D imaging?

Integrating ITGAX antibody staining with tissue clearing techniques presents unique challenges due to the membrane localization of CD11c and the potential for epitope destruction during clearing processes. A successful approach includes:

First, evaluate compatibility between the selected clearing method and ITGAX epitope preservation. Hydrogel-based techniques (CLARITY, PACT) or solvent-based methods (iDISCO, 3DISCO) may differentially affect epitope accessibility. Preliminary testing with each clearing protocol on small tissue sections can identify optimal methods before proceeding to valuable experimental samples.

Second, adjust antibody concentration and incubation times compared to standard immunofluorescence protocols. Tissue clearing typically requires higher antibody concentrations (2-5× standard concentrations) and extended incubation periods (often 3-7 days at 4°C with gentle agitation) to ensure adequate penetration into thick tissue sections. For ITGAX antibodies typically used at 1:500-1:2000 dilutions in standard IF , concentrations of 1:100-1:500 may be more appropriate for cleared tissue volumes.

Third, implement specific strategies to enhance antibody penetration. These include extending primary antibody incubation time, incorporating mild detergents (0.1-0.3% Triton X-100) in staining buffers, and potentially using fragment antibodies (Fab) when steric hindrance limits penetration into dense tissues.

Fourth, adapt mounting and imaging parameters for the cleared tissue's refractive index. Each clearing method creates specific optical properties that require matching mounting media and objective lenses. For instance, CLARITY-processed tissues have a refractive index of approximately 1.45, requiring appropriate immersion media and objectives for optimal ITGAX signal detection throughout the tissue volume.

What approaches can researchers use to quantify ITGAX expression in single-cell analysis techniques?

Quantifying ITGAX expression at the single-cell level requires specialized approaches for accurate data interpretation. Key methodological considerations include:

First, establish appropriate normalization standards for flow cytometry applications. Using calibration beads with known antibody binding capacity (ABC) allows conversion of arbitrary fluorescence units to molecules of equivalent soluble fluorochrome (MESF) or antibodies bound per cell (ABC). This standardization enables meaningful comparison between experiments, instruments, and research groups.

Second, implement rigorous gating strategies that account for autofluorescence and non-specific binding. ITGAX expression exists along a continuum in myeloid populations, making binary positive/negative gates potentially misleading. Fluorescence-minus-one (FMO) controls are particularly valuable for setting accurate gates for ITGAX+/- populations.

Third, utilize computational approaches for high-dimensional cytometry data. When analyzing ITGAX in the context of multiple markers (mass cytometry, spectral cytometry), unsupervised clustering algorithms (FlowSOM, PhenoGraph) or dimension reduction techniques (tSNE, UMAP) help identify cell populations based on their complete phenotypic profile rather than arbitrary gates on individual markers.

Fourth, validate flow cytometry findings with orthogonal techniques. Correlation of ITGAX quantification between flow cytometry and techniques like quantitative immunofluorescence microscopy, quantitative PCR, or proteomics provides greater confidence in expression measurements. For instance, ITGAX protein detected by Western blot at approximately 145 kDa should correlate with flow cytometry signal intensity across matched samples.

What are the most common causes of false-positive and false-negative results when using ITGAX antibodies?

Understanding the mechanisms behind false results enables researchers to implement appropriate controls and optimize protocols. Common causes include:

False-Positive Results:

• Cross-reactivity with structurally similar proteins, particularly other integrin family members. Validation using ITGAX-knockout controls or multiple antibody clones targeting different epitopes can identify cross-reactivity issues.

• Non-specific binding to Fc receptors on myeloid cells, which can be mitigated using Fc receptor blocking reagents before primary antibody incubation.

• Tissue autofluorescence, particularly in tissues rich in elastin, collagen, or lipofuscin. This can be addressed through autofluorescence quenching protocols or spectral unmixing during analysis.

False-Negative Results:

• Epitope masking during fixation. Different fixation methods (paraformaldehyde, methanol, acetone) variably preserve ITGAX epitopes. Optimization of fixation conditions or testing multiple antibody clones can overcome this issue.

• Ineffective antigen retrieval. ITGAX antibodies may require specific retrieval conditions, such as TE buffer pH 9.0 or citrate buffer pH 6.0 as recommended for the 60258-1-Ig clone .

• Protein degradation during sample preparation. ITGAX can be sensitive to proteolytic degradation, particularly in tissue samples with high protease activity. Inclusion of protease inhibitors during sample preparation can preserve epitope integrity.

To distinguish true signals from artifacts, researchers should implement biological controls (ITGAX-high and ITGAX-low cell populations), technical controls (isotype controls, secondary-only controls), and perform systematic protocol optimization.

How should ITGAX antibodies be stored and handled to maintain optimal performance?

Proper storage and handling of ITGAX antibodies is critical for maintaining their specificity and sensitivity. Key recommendations include:

First, follow manufacturer-specific guidelines for temperature conditions. Most ITGAX antibodies should be stored at -20°C for long-term preservation. For example, the 60258-1-Ig antibody is stable for one year when stored at -20°C . For short-term storage and frequent use, 4°C storage for up to one month is generally acceptable .

Second, minimize freeze-thaw cycles, which can cause antibody denaturation and aggregation. Aliquoting antibodies into single-use volumes upon receipt is recommended, though some formulations (like those containing 50% glycerol) may be less sensitive to freeze-thaw damage and manufacturers may indicate that "aliquoting is unnecessary for -20°C storage" .

Third, pay attention to buffer composition. Many ITGAX antibodies are supplied in PBS with preservatives like 0.02% sodium azide and stabilizers like 50% glycerol . These components help maintain antibody functionality during storage and should not be diluted unless immediately before use.

Fourth, implement quality control testing for antibodies in long-term storage. Periodic validation using positive control samples (such as THP-1 cells, HL-60 cells, or U-937 cells for ITGAX) can confirm that stored antibodies retain their specific reactivity before use in critical experiments.

Finally, maintain proper documentation of antibody performance over time. Recording lot numbers, dates of receipt, aliquoting, and validation testing helps track potential variability and troubleshoot inconsistent results.

What controls are essential when using ITGAX antibodies for quantitative analysis in different experimental systems?

Rigorous control strategies are essential for generating reliable quantitative data with ITGAX antibodies. The following controls should be implemented:

Biological Controls:

• Positive control samples with known ITGAX expression, such as human spleen tissue, THP-1 cells, HL-60 cells, or U-937 cells .

• Negative control samples lacking ITGAX expression, ideally including genetic knockouts or knockdowns of ITGAX when available.

• Titration series of samples with graded ITGAX expression levels to establish the dynamic range and linearity of detection.

Technical Controls:

• Isotype controls using non-specific antibodies of the same isotype, host species, and concentration as the ITGAX antibody. For instance, mouse IgG2a isotype controls would be appropriate for the 60258-1-Ig antibody .

• Secondary antibody-only controls to assess background from the detection system.

• Blocking peptide controls using the immunizing peptide to confirm signal specificity.

Quantification Controls:

• Standard curves using recombinant ITGAX protein or calibrator cells with known ITGAX expression levels.

• Internal reference controls (housekeeping proteins for Western blot, invariant cellular markers for flow cytometry) to normalize ITGAX signals.

• Technical replicates to assess method precision and biological replicates to assess natural variation.

When comparing ITGAX expression across different experimental conditions, standardized protocols for sample collection, processing, and analysis are crucial. Batch effects should be minimized by processing comparable samples simultaneously or including shared reference samples across batches.

How can researchers utilize ITGAX antibodies in single-cell spatial transcriptomics studies?

Integrating protein-level ITGAX detection with spatial transcriptomics represents an emerging frontier in immunology research. Methodological approaches include:

First, implement sequential immunofluorescence and in situ hybridization protocols. ITGAX protein can be detected using immunofluorescence with validated antibodies (such as 60258-1-Ig at 1:500-1:2000 dilution) , followed by in situ hybridization for ITGAX mRNA and other transcripts of interest. Careful optimization of fixation conditions is essential to preserve both epitopes for antibody binding and nucleic acids for hybridization.

Second, explore commercial platforms that combine protein and RNA detection. Technologies like NanoString GeoMx DSP or 10x Genomics Visium with antibody capture can simultaneously detect ITGAX protein and gene expression signatures in spatial context. These approaches require specialized antibody conjugates compatible with the respective platforms.

Third, develop computational frameworks for integrating protein and transcriptional data. Correlation analyses between ITGAX protein levels (from immunofluorescence) and mRNA expression (from spatial transcriptomics) can reveal post-transcriptional regulation mechanisms. Cell segmentation algorithms that delineate individual cells in tissue contexts enable single-cell resolution for these multi-modal analyses.

Fourth, implement experimental designs that capture dynamic processes. Sequential tissue sections analyzed for ITGAX protein and mRNA at different timepoints can reveal temporal relationships between transcriptional regulation and protein expression during immune responses or disease progression.

What are the considerations for using ITGAX antibodies in studying extracellular vesicles and exosomes?

Detecting ITGAX on extracellular vesicles (EVs) presents unique technical challenges that require specialized approaches:

First, optimize immunocapture strategies for ITGAX-positive EVs. Antibodies like the 8E3 clone or ITGAX/1284 can be conjugated to beads or plates for capturing EVs expressing CD11c. Critical variables include antibody orientation, conjugation chemistry, and blocking conditions to minimize non-specific binding while maintaining epitope accessibility.

Second, implement rigorous EV isolation and purification protocols. Differential ultracentrifugation, size exclusion chromatography, or commercial EV isolation kits should be validated specifically for preserving ITGAX epitopes. The membrane localization of CD11c may make it susceptible to damage during harsh isolation procedures.

Third, develop sensitive detection methods for ITGAX on individual EVs. Given the small size of exosomes (30-150 nm), techniques like high-resolution flow cytometry with fluorescence amplification, surface plasmon resonance, or super-resolution microscopy may be required to reliably detect ITGAX on individual vesicles.

Fourth, validate EV findings with complementary approaches. ITGAX detection on EVs should be confirmed using multiple techniques, such as immunoblotting of EV lysates (where ITGAX should appear at approximately 145 kDa) , immuno-electron microscopy for direct visualization, and correlation with proteomics data.

Fifth, implement appropriate controls specific to EV studies. These include EVs from ITGAX-knockout or knockdown cells, detergent treatments to distinguish membrane-bound signals from protein aggregates, and density gradient separation to confirm association with bona fide EVs rather than protein complexes.

How can researchers leverage ITGAX antibodies in developing new immunotherapeutic approaches?

ITGAX antibodies have significant potential in developing targeted immunotherapies, particularly for manipulating dendritic cell and macrophage functions. Key research applications include:

First, develop and validate targeting strategies for CD11c-positive cells. Antibodies like the N418 clone, available in functional grade preparations suitable for in vivo applications , can be used to deliver payloads specifically to CD11c-expressing cells. These targeting approaches require rigorous validation of antibody specificity, internalization efficiency, and payload release kinetics.

Second, engineer bispecific or multispecific antibodies incorporating anti-ITGAX domains. These complex biologics can simultaneously bind CD11c and other relevant targets (e.g., tumor antigens, pathogen components, or T cell markers) to promote specific immune interactions. Careful epitope mapping and binding kinetics analysis ensure that the ITGAX-binding domain maintains specificity and affinity within the multispecific construct.

Third, investigate the functional consequences of ITGAX engagement. Beyond targeting, some antibodies may modulate CD11c function, potentially altering adhesion, migration, or signaling in myeloid cells. Functional assays measuring phagocytosis, cytokine production, and T cell activation can characterize these effects and identify therapeutic applications.

Fourth, develop comprehensive characterization panels for monitoring CD11c-positive populations during immunotherapy. Multiplex flow cytometry or mass cytometry panels incorporating ITGAX antibodies alongside other myeloid and activation markers can track therapy-induced changes in dendritic cell and macrophage populations. These monitoring strategies require standardized protocols with appropriate compensation and calibration controls.