STAB2 Antibody

What is Stabilin-2 and why is it important in research?

Stabilin-2 is a large transmembrane receptor protein (277 kDa) encoded by the STAB2 gene in humans. It consists of 2551 amino acids and functions as a major scavenger receptor, particularly for hyaluronic acid (HA) clearance from circulation . Also known by alternative names including FEEL2, FELE-2, FELL2, and FAS1 EGF-like and X-link domain containing adhesion molecule-2, Stabilin-2 plays crucial roles in maintaining vascular homeostasis by regulating the uptake and clearance of various extracellular molecules including lipoproteins, advanced glycation end products, and apoptotic cells . Research interest in Stabilin-2 has grown significantly due to its implications in pathological conditions such as atherosclerosis, inflammation, and cancer metastasis .

What applications are STAB2 antibodies validated for?

STAB2 antibodies have been validated for multiple experimental applications depending on the specific antibody clone and manufacturer. Common applications include:

• Western Blot (WB) for protein expression analysis

• Enzyme-Linked Immunosorbent Assay (ELISA) for quantitative detection

• Immunohistochemistry (IHC) for tissue localization studies

• Immunocytochemistry (ICC) and Immunofluorescence (IF) for cellular localization

• Immunoprecipitation (IP) for protein-protein interaction studies

When selecting a STAB2 antibody, researchers should verify the specific applications for which each antibody has been validated, as performance can vary significantly between different clones and suppliers .

What species reactivity is available for STAB2 antibodies?

STAB2 antibodies are available with reactivity to multiple species, most commonly:

• Human

• Mouse

• Rat

Some antibodies also demonstrate cross-reactivity with canine, porcine, and monkey Stabilin-2 . For specific research applications, it's crucial to select an antibody with validated reactivity to your species of interest. The sequence homology between species should be considered when antibody cross-reactivity is important for comparative studies .

How should STAB2 antibodies be stored and handled to maintain activity?

For optimal performance and longevity of STAB2 antibodies:

• Store unconjugated antibodies at -20°C for long-term storage

• Avoid repeated freeze-thaw cycles by aliquoting antibodies upon receipt

• For short-term use (1-2 weeks), store at 4°C

• Conjugated antibodies (e.g., FITC, HRP) may have specific storage requirements (often 4°C protected from light)

• Follow manufacturer-specific recommendations for dilution buffers

• Use appropriate blocking reagents to minimize non-specific binding

• Include proper positive and negative controls in each experiment

How can STAB2 antibodies be used to study cancer metastasis mechanisms?

Research has demonstrated that Stabilin-2 plays a significant role in cancer metastasis, making STAB2 antibodies valuable tools for investigating these mechanisms:

• Blocking experiments: Anti-STAB2 blocking antibodies can be administered in animal models to evaluate the effect of Stabilin-2 inhibition on tumor metastasis. Studies have shown that administration of anti-STAB2 antibody in wild-type mice elevated circulating hyaluronic acid (HA) levels and prevented tumor metastasis .

• Mechanistic studies: STAB2 antibodies can help investigate the relationship between Stabilin-2, circulating HA levels, and tumor cell attachment to vascular endothelium. Research has shown that elevated circulating HA (resulting from Stabilin-2 inhibition) inhibits the rolling/tethering of tumor cells to lung endothelial cells .

• Immunohistochemical analysis: STAB2 antibodies can be used to evaluate Stabilin-2 expression in different tissue types and correlate expression patterns with metastatic potential .

The experimental approach should include appropriate controls and combine molecular and cellular techniques to comprehensively investigate the role of Stabilin-2 in metastasis.

What controls should be included when using STAB2 antibodies in immunohistochemistry?

For rigorous immunohistochemistry experiments with STAB2 antibodies, the following controls are essential:

• Positive tissue control: Use tissues known to express Stabilin-2, such as liver sinusoidal endothelial cells. This control validates that the staining protocol is working correctly .

• Negative tissue control: Include tissues known to be STAB2-negative, processed identically to experimental samples. This helps verify antibody specificity and identify any background staining issues .

• Internal negative controls: Utilize various cell types within the same tissue section that do not express Stabilin-2 as internal negative controls .

• Isotype control: Include an irrelevant antibody of the same isotype, host species, and concentration as the STAB2 antibody to identify any non-specific binding due to the antibody class.

• Absorption control: Pre-incubate the STAB2 antibody with purified Stabilin-2 protein to confirm binding specificity.

Additionally, it's recommended to validate results using multiple STAB2 antibody clones when possible, particularly when studying tissues with potentially variable expression levels.

How do STAB2 knockout models and anti-STAB2 antibody administration differ in research applications?

Both STAB2 knockout models and anti-STAB2 antibody administration are valuable research tools, but they offer different experimental advantages:

Research has shown that both approaches can significantly elevate circulating HA levels and prevent tumor metastasis, suggesting that functional inhibition of Stabilin-2 may be a potential strategy to suppress tumor metastasis .

What methodological considerations are important when using STAB2 antibodies to analyze hyaluronic acid clearance?

When investigating Stabilin-2's role in hyaluronic acid clearance using antibodies:

• Timing considerations: Measure serum HA levels at multiple timepoints after anti-STAB2 antibody administration. Research has shown that serum HA levels increase within 3 days of anti-STAB2 mAb injection in mouse models .

• Antibody selection: Choose antibodies that specifically block the HA-binding domain of Stabilin-2. In vitro assays can verify an antibody's ability to inhibit HA binding to Stabilin-2, such as measuring internalization of FITC-labeled HA in hepatic sinusoidal endothelial cells (HSECs) .

• Dosage optimization: Titrate antibody concentrations to determine optimal dosing for achieving desired HA elevation without off-target effects. In previous studies, intraperitoneal injections every 3 days proved effective .

• Measurement techniques: Use validated assays for measuring serum HA levels, such as enzyme-linked immunosorbent assays specific for HA.

• Control experiments: Include parallel experiments with control IgG of the same isotype to confirm effects are specific to Stabilin-2 blockade .

• Alternative approaches: Consider complementary techniques such as competitive binding assays or surface plasmon resonance to characterize antibody-Stabilin-2 interactions.

How can non-specific binding be minimized when working with STAB2 antibodies?

Non-specific binding is a common challenge when working with STAB2 antibodies. To minimize this issue:

• Optimize blocking protocol: Use a protein blocking solution appropriate for your sample type (e.g., 5-10% normal serum from the same species as the secondary antibody, BSA, or commercial blocking solutions).

• Antibody titration: Perform careful titration experiments to determine the optimal antibody concentration that maximizes specific signal while minimizing background.

• Incubation conditions: Adjust incubation temperature and duration; overnight incubation at 4°C often reduces non-specific binding compared to shorter incubations at room temperature.

• Washing steps: Increase the number and duration of washing steps using buffers containing mild detergents like 0.05-0.1% Tween-20.

• Secondary antibody selection: Use highly cross-adsorbed secondary antibodies to reduce cross-reactivity with endogenous immunoglobulins.

• Endogenous peroxidase/phosphatase quenching: For IHC applications, properly quench endogenous enzymes that may react with detection substrates.

• Consider monoclonal alternatives: If using polyclonal antibodies with high background, switching to a monoclonal antibody might improve specificity .

What approaches can resolve contradictory results when studying Stabilin-2 expression using different antibodies?

When faced with contradictory results using different STAB2 antibodies:

• Epitope mapping: Determine the specific epitopes recognized by each antibody. Antibodies targeting different domains of Stabilin-2 may yield varying results, especially if certain domains are masked in different tissue contexts.

• Multiple detection methods: Validate expression using complementary techniques such as Western blot, IHC, and qPCR to confirm protein presence and localization.

• Knockout/knockdown validation: Use STAB2 knockout tissues or knockdown cells as negative controls to confirm antibody specificity.

• Isoform awareness: Consider whether antibodies detect different STAB2 isoforms or post-translationally modified variants.

• Context-dependent expression: Evaluate whether expression differences reflect biological variability rather than technical issues.

• Sample preparation differences: Standardize fixation, antigen retrieval, and processing methods across experiments.

• Cross-reactivity analysis: Test for potential cross-reactivity with Stabilin-1 or other related proteins that share structural similarities.

What are the optimal fixation and antigen retrieval methods for STAB2 immunohistochemistry?

Successful STAB2 immunohistochemistry depends on proper fixation and antigen retrieval:

• Fixation options:

  • Formalin fixation (10% neutral buffered formalin for 24-48 hours) is commonly used

  • Cold acetone fixation (10 minutes) may preserve certain epitopes better for frozen sections

  • Paraformaldehyde (4%) can be suitable for immunofluorescence applications

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) at 95-100°C for 20 minutes

  • For some antibody clones, EDTA buffer (pH 9.0) may yield better results

  • Enzymatic retrieval using proteinase K can be effective but requires careful optimization

• Optimization considerations:

  • Test multiple retrieval methods with each new antibody or tissue type

  • Adjust retrieval time based on fixation duration (longer fixation typically requires more aggressive retrieval)

  • Include positive control tissues with known Stabilin-2 expression to validate protocol effectiveness

Different antibody clones may have specific recommendations for optimal detection - always refer to manufacturer protocols as a starting point.

How can STAB2 antibodies be utilized in the study of hyaluronic acid-based therapeutic approaches?

Emerging research applications for STAB2 antibodies in hyaluronic acid-based therapeutics include:

• Extending HA therapeutic half-life: By temporarily blocking Stabilin-2 function with antibodies, researchers can prolong the circulation time of HA-based therapeutics, potentially enhancing their efficacy .

• Tumor metastasis prevention: Research has demonstrated that administration of anti-STAB2 antibodies elevates serum HA levels and prevents tumor metastasis. This approach could be developed into potential anti-metastatic therapies .

• Combinatorial approaches: STAB2 antibodies might be used in combination with HA-conjugated drugs to improve drug delivery by increasing circulation time and reducing hepatic clearance.

• Mechanistic investigations: STAB2 antibodies can help elucidate the molecular mechanisms by which elevated circulating HA inhibits tumor cell attachment to vascular endothelium, potentially informing new therapeutic strategies .

• Biomarker development: By understanding the relationship between Stabilin-2 function and HA metabolism, researchers can potentially develop biomarkers for disease progression or treatment response.

Experimental approaches should carefully monitor both intended effects on HA levels and potential off-target consequences of altering this important scavenging pathway.

What is the significance of studying Stabilin-2 in the context of inflammatory responses?

The role of Stabilin-2 in inflammatory processes represents an important research area:

• Clearance of damage-associated molecular patterns: Stabilin-2 functions in clearing apoptotic cells and cellular debris, which is critical for resolving inflammation and preventing autoimmune responses.

• Regulation of leukocyte trafficking: Research suggests Stabilin-2 may play a role in leukocyte adhesion and migration during inflammatory responses.

• HA-mediated inflammation modulation: By regulating circulating HA levels, Stabilin-2 may indirectly influence inflammatory processes, as HA fragments can have pro-inflammatory effects .

• Experimental approaches: STAB2 antibodies can be used to:

  • Block Stabilin-2 function in inflammatory disease models

  • Assess Stabilin-2 expression patterns in inflamed tissues

  • Investigate the relationship between Stabilin-2 expression and inflammatory biomarkers

• Potential therapeutic applications: Understanding Stabilin-2's role in inflammation could lead to novel anti-inflammatory strategies targeting this receptor or its signaling pathways.

Research protocols should incorporate both acute and chronic inflammatory models to fully characterize the temporal aspects of Stabilin-2's role in inflammation resolution.

How might STAB2 antibodies contribute to the development of anti-metastatic therapies?

The potential of STAB2 antibodies in anti-metastatic therapy development is supported by several research findings and could be explored through:

• Therapeutic antibody optimization: Developing humanized or fully human anti-STAB2 antibodies with optimized pharmacokinetic properties for potential clinical applications.

• Combination therapy approaches: Investigating synergistic effects between STAB2 antibodies and conventional chemotherapies or immunotherapies. Studies have shown that anti-STAB2 antibody administration prevented the metastasis of not only mouse melanoma cells but also human breast tumor cells without notable side effects .

• Cancer-specific targeting: Exploring whether certain cancer types are particularly susceptible to anti-metastatic effects of STAB2 inhibition. Research has demonstrated efficacy against melanoma and breast cancer metastasis models .

• Mechanism elucidation: Further investigating how elevated HA levels inhibit tumor cell attachment to endothelium, including the specific receptors and signaling pathways involved.

• Predictive biomarkers: Identifying biomarkers that could predict which patients might benefit most from anti-STAB2 therapeutic approaches.

The finding that Stabilin-2 knockout mice displayed no overt developmental defects despite dramatically increased plasma HA levels suggests that therapeutic targeting of this pathway might have a favorable safety profile .

What are the methodological considerations for developing function-blocking STAB2 antibodies?

Developing effective function-blocking STAB2 antibodies requires careful consideration of several factors:

• Epitope selection: Target the HA-binding domains of Stabilin-2 to specifically block HA clearance. The Link domain and X-Link domain are critical for HA recognition.

• Screening assays: Establish robust functional screening assays such as:

  • HA internalization assays using hepatic sinusoidal endothelial cells

  • Competitive binding assays with labeled HA

  • Cell-based receptor occupancy assays

• Antibody format optimization: Consider different antibody formats (full IgG, Fab, scFv) depending on the intended application and required tissue penetration.

• Validation methods:

  • In vitro: Demonstrate inhibition of HA binding to Stabilin-2 expressing cells

  • In vivo: Confirm elevation of serum HA levels after antibody administration

  • Functional: Verify biological effects such as inhibition of tumor cell adhesion

• Species cross-reactivity: When developing therapeutic candidates, consider antibodies that cross-react with both human and relevant animal models to facilitate translational research.

• Manufacturing considerations: Optimize expression systems and purification protocols to ensure consistent antibody quality and function.