LYVE1 Antibody

What is LYVE1 and why is it important as a research target?

LYVE1 is a transmembrane glycoprotein receptor for the extracellular matrix mucopolysaccharide hyaluronan (HA). It shares structural similarity with CD44, containing a conserved hyaluronan binding domain in its extracellular region. LYVE1 serves as one of the principal markers for lymphatic endothelial cells (LECs), alongside podoplanin, PROX-1, Tie-2, and VEGFR-3. Its importance as a research target stems from its relatively specific expression pattern on lymphatic vessels, making it invaluable for studying lymphatic system development, function, and pathology. The lymphatic system plays crucial roles in immune surveillance, protein transport, and cellular trafficking throughout the body, particularly for dendritic cells . LYVE1 expression has also been documented in certain populations of macrophages and liver sinusoidal endothelial cells, indicating its broader functional significance beyond just lymphatic vessels .

What is the expression pattern of LYVE1 across different tissues and species?

LYVE1 expression demonstrates a consistent pattern across species, though with some notable variations:

In humans:

• Prominently expressed in lymphatic endothelial cells of various tissues

• Detected in tonsil lymphatic vessels through immunohistochemistry

• Present in liver and spleen tissues as confirmed by Western blot (approximately 60 kDa)

• Expressed in some populations of human macrophages, particularly those differentiated with M-CSF

• Found in human meninges, where cells co-express multiple lymphatic endothelial cell markers

In mice:

• Expressed in lymphatic vessels across multiple tissues

• Strongly detected in liver sinusoidal endothelial cells

• Present in mouse meninges during embryonic development (E12.5, E13.5) and continues through E18, where it co-expresses with PROX1 and MRC1

• Detected in mouse lung and heart tissue

• Appears in peritumoral lymphatic endothelial cells in various tumor models (B16F10, E0771, MMTV-PyMT)

Interestingly, the expression intensity and pattern can vary under pathological conditions, with studies showing upregulation of MHC-II and PD-L1 in peritumoral lymphatic endothelial cells compared to naïve dermal LECs .

What criteria should I use when selecting an appropriate LYVE1 antibody for my experiments?

Selecting the appropriate LYVE1 antibody requires careful consideration of several factors:

• Species reactivity: Ensure the antibody recognizes LYVE1 in your species of interest. The search results show antibodies specific for human LYVE1 (e.g., AF2089, MAB20892) and mouse LYVE1 (e.g., AF2125, ALY7) .

• Application compatibility: Verify the antibody has been validated for your intended application:

  • For Western blot: Consider antibodies demonstrated to detect the appropriate size band (60-70 kDa for LYVE1)

  • For IHC: Select antibodies shown to work in your specific fixation method (paraffin vs. frozen sections)

  • For flow cytometry: Choose antibodies validated in suspension with appropriate cellular models

• Clonality: Monoclonal antibodies offer consistency between lots but may be sensitive to epitope masking, while polyclonal antibodies provide broader epitope recognition but potential lot-to-lot variability.

• Format: Consider whether you need unconjugated antibodies (for flexible detection strategies) or directly conjugated antibodies (for multicolor flow cytometry or direct visualization) .

• Validation data quality: Examine the provided validation data thoroughly. Robust antibodies should show clear, specific staining patterns consistent with known LYVE1 biology across multiple validation methods .

• Published literature: Search for papers that have successfully used the antibody in applications similar to yours, particularly those studying similar biological questions.

How can I validate a LYVE1 antibody for specificity in my experimental system?

Validating LYVE1 antibody specificity is critical for reliable results. A comprehensive validation approach should include:

• Positive and negative tissue controls:

  • Positive controls: Use tissues known to express LYVE1 such as lymph nodes, tonsil, liver sinusoids, or spleen

  • Negative controls: Include tissues with minimal LYVE1 expression or use isotype control antibodies to assess non-specific binding

• Western blot validation:

  • Confirm a single specific band at the expected molecular weight (approximately 60-70 kDa for LYVE1)

  • Include both positive control lysates (lymphatic endothelial cells, liver tissue) and negative control lysates

• RNAi or knockout confirmation:

  • Use siRNA/shRNA against LYVE1 or CRISPR-based LYVE1 knockout models to confirm signal reduction/loss

• Co-staining with alternative LYVE1 antibodies:

  • Use antibodies from different hosts or against different epitopes to verify staining patterns

• Multi-marker co-expression:

  • Confirm co-expression with other lymphatic markers like Prox1, podoplanin, or VEGFR-3 in lymphatic endothelium

  • The search results show examples of co-staining of LYVE1 with PROX1 and MRC1 in mouse meninges

• Blocking peptide competition:

  • Pre-incubate the antibody with the immunizing peptide to demonstrate specific binding

• Flow cytometry validation:

  • Compare staining in cell types known to express LYVE1 (e.g., lymphatic endothelial cells) versus negative cells

  • Include appropriate isotype controls as demonstrated in the HUVEC cells staining example

What are the optimal conditions for detecting LYVE1 in Western blot applications?

Optimal conditions for LYVE1 detection by Western blot require careful attention to sample preparation, running conditions, and detection parameters:

• Sample preparation:

  • Use tissues with known LYVE1 expression such as liver, spleen, or cultured lymphatic endothelial cells

  • Employ RIPA or similar lysis buffers containing protease inhibitors to preserve protein integrity

  • The search results indicate successful detection in human liver and spleen tissue lysates, as well as in cell lines including HeLa, MCF-7, and 293T

• Electrophoresis conditions:

  • Run samples under reducing conditions using standard SDS-PAGE protocols

  • Use Immunoblot Buffer Group 1 or 8 as specified in several successful protocols

  • Load sufficient protein (typically 20-50 μg total protein per lane)

• Antibody concentration and incubation:

  • For Human LYVE1: Use AF2089 at 0.25-1 μg/mL or MAB20892 at 1 μg/mL

  • For Mouse LYVE1: Use AF2125 at 0.25 μg/mL

  • Incubate primary antibodies overnight at 4°C for optimal binding

• Detection system:

  • Use appropriate HRP-conjugated secondary antibodies (e.g., Anti-Goat IgG for AF2089 and AF2125, Anti-Mouse IgG for MAB20892)

  • Employ enhanced chemiluminescence (ECL) for sensitive detection

• Expected results:

  • Human LYVE1: Expect a band at approximately 60-70 kDa

  • Mouse LYVE1: Expect a band at approximately 60-65 kDa

  • Note that glycosylation may cause molecular weight variations across different cell/tissue types

• Special considerations:

  • TGF-beta treatment has been shown to reduce LYVE1 expression in LECs, which could be used as a negative control

  • Including positive control lysates (e.g., liver tissue) alongside experimental samples helps validate successful detection

What protocol modifications are recommended for optimal LYVE1 immunohistochemistry in different tissue types?

LYVE1 immunohistochemistry requires specific protocol modifications based on tissue type, fixation method, and detection system:

• Fixation considerations:

  • Paraffin-embedded tissues: 4% paraformaldehyde fixation works well, though epitope retrieval is typically necessary

  • Frozen sections: Generally provide better epitope preservation and may require less stringent retrieval methods

  • Perfusion-fixed tissues (for animal models): Often yield superior results for vascular markers like LYVE1

• Tissue-specific protocols:

  • Human tonsil: 15 μg/mL of AF2089 (Goat Anti-Human LYVE1) with overnight incubation at 4°C works effectively

  • Mouse liver: 15 μg/mL of AF2125 (Goat Anti-Mouse LYVE1) with overnight incubation at 4°C yields specific labeling of sinusoidal endothelial cells

  • Mouse lung and heart: Paraffin-embedded sections have been successfully stained using the RM0033-4D17 antibody

  • Mouse and human meninges: Co-staining protocols with PROX1 and MRC1 have been established

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • For paraffin sections, more aggressive retrieval may be necessary compared to frozen sections

• Detection systems:

  • Chromogenic detection: Anti-Goat HRP-DAB Cell & Tissue Staining Kit has been successfully used

  • Fluorescent detection: Appropriate fluorophore-conjugated secondary antibodies for multicolor imaging

  • Counterstaining: Hematoxylin works well as a nuclear counterstain with chromogenic detection

• Special considerations:

  • Background reduction: Include a blocking step with serum from the same species as the secondary antibody

  • Autofluorescence mitigation: For fluorescent detection in tissues with high autofluorescence (e.g., liver), consider using Sudan Black B treatment or spectral unmixing

  • For co-staining: Carefully plan antibody combinations to avoid cross-reactivity; sequential staining may be necessary

How should I interpret variations in LYVE1 expression levels across different pathological conditions?

Interpreting variations in LYVE1 expression across pathological conditions requires careful consideration of multiple factors:

• Baseline expression understanding:

  • Normal LYVE1 expression is predominantly in lymphatic endothelial cells, liver sinusoidal cells, and some macrophage populations

  • Expression intensity can vary naturally between different anatomical locations even in healthy tissues

• Cancer-associated changes:

  • Peritumoral lymphatic vessels often show altered LYVE1 expression compared to normal lymphatics

  • Evidence shows that peritumoral LECs upregulate MHC-II and PD-L1 compared to naïve dermal LECs in multiple tumor models (B16F10, E0771, MMTV-PyMT)

  • These changes may reflect functional alterations in tumor-associated lymphatics, potentially related to immune regulation and metastasis

• Inflammatory conditions:

  • TGF-beta (1, 2, and 3) treatment reduces LYVE1 expression in lymphatic endothelial cells, suggesting inflammation-mediated regulation

  • This downregulation occurs alongside changes in other lymphatic markers like Prox-1 and VEGFR-3

• Developmental context:

  • LYVE1 expression changes during embryonic development, as seen in mouse meninges from E12.5 through E18

  • These changes may reflect normal developmental processes versus pathological alterations

• Aging and neurodegeneration:

  • LYVE1 expression has been detected in human meninges both in samples without neuropathology and in elderly brains with evidence of neuropathology

  • Careful comparison with age-matched controls is essential when studying neurodegenerative conditions

• Technical considerations for interpretation:

  • Always normalize expression to appropriate housekeeping proteins/genes

  • Use quantitative methods (densitometry for Western blots, quantitative image analysis for IHC)

  • Compare multiple timepoints when studying progressive conditions

  • Consider using multiple detection methods (e.g., Western blot and IHC) to validate findings

What are common technical challenges when working with LYVE1 antibodies and how can they be overcome?

Researchers frequently encounter several technical challenges when working with LYVE1 antibodies. Here are the most common issues and their solutions:

• Weak or absent signal:

  • Cause: Insufficient antibody concentration, epitope masking, or low target expression

  • Solution: Optimize antibody concentration (try 0.25-1 μg/mL for Western blot , 15 μg/mL for IHC ), enhance antigen retrieval methods, and use tissues/cells with known LYVE1 expression as positive controls

• Non-specific binding and high background:

  • Cause: Insufficient blocking, excessive antibody concentration, or cross-reactivity

  • Solution: Increase blocking time/concentration, optimize antibody dilution, include proper negative and isotype controls , and use more stringent washing steps

• Inconsistent results between experiments:

  • Cause: Variations in sample preparation, antibody lots, or detection methods

  • Solution: Standardize protocols, include consistent positive controls across experiments, and consider using monoclonal antibodies for greater consistency

• Discrepancies between detection methods:

  • Cause: Different epitope accessibility in various applications (WB vs. IHC vs. flow cytometry)

  • Solution: Validate antibodies separately for each application, and be aware that some antibodies may work well for one application but not others

• Glycosylation-related issues:

  • Cause: LYVE1 is heavily glycosylated, causing molecular weight variations and potential epitope masking

  • Solution: For Western blots, consider treating samples with glycosidases if needed; for IHC/flow cytometry, be aware that epitope masking by sialylated O-linked glycan chains may occur in vivo

• Fixation artifacts in IHC:

  • Cause: Over-fixation or inappropriate fixative choice can mask LYVE1 epitopes

  • Solution: Optimize fixation protocols (duration, fixative type), compare frozen versus paraffin-embedded sections, and test different antigen retrieval methods

• Co-staining challenges:

  • Cause: Antibody cross-reactivity or incompatible detection systems

  • Solution: Use antibodies raised in different host species, employ sequential staining protocols, and carefully select compatible fluorophores to avoid spectral overlap

How can LYVE1 antibodies be used to investigate tumor-associated lymphangiogenesis and metastasis?

LYVE1 antibodies serve as powerful tools for investigating tumor-associated lymphangiogenesis and metastasis through multiple sophisticated approaches:

• Characterization of peritumoral lymphatic vessels:

  • LYVE1 antibodies allow visualization and quantification of lymphatic vessel density around tumors

  • Research has shown that peritumoral lymphatic vessels display altered phenotypes, with upregulation of MHC-II, PD-L1, PD-L2, HVEM, and CD48 compared to normal lymphatics

  • These changes were documented in multiple tumor models including B16F10 melanoma, E0771 mammary carcinoma, and MMTV-PyMT spontaneous mammary tumors

  • Quantitative assessment can be performed using parameters such as vessel density, diameter, and branching

• Temporal monitoring of lymphangiogenic processes:

  • Using LYVE1 antibodies across different timepoints (e.g., day 7, 14, and 21 post-tumor inoculation) allows tracking of lymphangiogenesis progression

  • This approach enables correlation between lymphatic vessel changes and tumor progression stages

• Molecular profiling of tumor-associated lymphatics:

  • Multi-color immunofluorescence combining LYVE1 with other markers (MHC-II, PD-L1, etc.) reveals immunoregulatory phenotype changes in tumor-associated lymphatics

  • This multi-dimensional molecular profiling approach can identify potential therapeutic targets on lymphatic vessels

• Functional assessment of lymphatic vessels in metastasis:

  • LYVE1 antibodies can help identify tumor cells within lymphatic vessels (lymphovascular invasion)

  • Coupling LYVE1 staining with tumor cell tracking enables visualization of metastatic routes

• Investigation of cytokine effects on lymphatic endothelium:

  • Research has shown that TGF-beta treatment reduces LYVE1 expression in lymphatic endothelial cells

  • This system allows investigation of how tumor-derived factors modify lymphatic vessel phenotype and function

• Methodological considerations for tumor studies:

  • Use both xenograft/syngeneic models and spontaneous tumor models for comprehensive assessment

  • Include analysis of both peritumoral and intratumoral lymphatics

  • Correlate lymphatic vessel parameters with metastatic burden in downstream lymph nodes

What innovative approaches combine LYVE1 antibodies with other technologies for advanced lymphatic system research?

Innovative research approaches are increasingly combining LYVE1 antibodies with cutting-edge technologies to advance lymphatic system research:

• Multi-dimensional Microscopic Molecular Profiling (MMMP):

  • This advanced approach combines iterative antibody staining and imaging cycles with computational integration

  • LYVE1 antibodies have been incorporated into MMMP workflows to map lymphatic networks in complex tissue contexts

  • The technique involves repeated cycles of staining, imaging, chemical bleaching, and re-staining the same tissue section

  • This enables simultaneous visualization of LYVE1 with dozens of other markers on the same tissue section

• Intravital imaging with LYVE1 antibodies:

  • Direct conjugation of LYVE1 antibodies with fluorophores suitable for in vivo imaging

  • Allows real-time visualization of lymphatic vessel function and cellular trafficking

  • Can be combined with fluorescently labeled tumor cells or immune cells to track interactions with lymphatics

• Single-cell omics integration:

  • LYVE1 antibodies can be used for cell sorting (FACS) of lymphatic endothelial cells prior to single-cell RNA-seq

  • This approach enables comprehensive transcriptomic profiling of lymphatic endothelial heterogeneity

  • Integration of protein expression data (from LYVE1 staining) with transcriptomic data provides multi-omic insights

• 3D reconstruction and computational modeling:

  • Serial section staining with LYVE1 antibodies allows 3D reconstruction of lymphatic networks

  • Computational approaches can then model fluid flow, cellular trafficking, and network connectivity

  • These models provide insights into lymphatic function that cannot be observed with standard 2D imaging

• Optogenetic and biosensor integration:

  • LYVE1 promoter-driven expression of optogenetic tools or biosensors

  • Enables functional manipulation and monitoring of lymphatic endothelial cells

  • Can be combined with LYVE1 antibody staining to correlate functional changes with spatial information

• LYVE1-targeted nanoparticle delivery systems:

  • LYVE1 antibodies can be conjugated to nanoparticles for targeted delivery to lymphatic vessels

  • Applications include lymphatic-specific drug delivery and molecular imaging

  • These approaches hold potential for both research and therapeutic applications

How does LYVE1 antibody performance compare with other lymphatic endothelial cell markers?

When designing lymphatic vessel research, understanding the comparative performance of different markers is essential:

Research has demonstrated that combining multiple markers provides the most robust identification of lymphatic vessels. For example, co-staining of LYVE1 with PROX1 and MRC1 has been effectively used to identify lymphatic-like cells in the meninges . The comparative analysis across multiple markers helps overcome the limitations of any single marker.

How should researchers address discrepancies in LYVE1 detection between different experimental platforms?

Researchers frequently encounter discrepancies in LYVE1 detection across different experimental platforms. Addressing these discrepancies requires systematic troubleshooting:

• Antibody epitope considerations:

  • Different antibodies target different LYVE1 epitopes, which may be differentially accessible depending on the technique

  • For human LYVE1, antibodies like AF2089 target the Ser24-Thr238 region , while for mouse LYVE1, antibodies like AF2125 target the Ala24-Thr234 region

  • Some epitopes may be masked by protein folding or post-translational modifications in certain applications

• Western blot vs. immunostaining discrepancies:

  • Denaturing conditions in Western blot may expose epitopes hidden in native conformation

  • Apparent molecular weight in Western blot (60-70 kDa) may differ from predicted weight due to glycosylation

  • Consider using multiple antibodies targeting different epitopes for confirmation across platforms

• Flow cytometry considerations:

  • Surface accessibility of LYVE1 may differ between fixed tissues and cells in suspension

  • Demonstrated protocols for flow cytometry include staining of HUVEC cells and PBMC-derived macrophages

  • Include appropriate isotype controls to establish specific staining thresholds

• Post-translational modification effects:

  • LYVE1 binding to its ligand hyaluronan in vivo may be masked by sialylated O-linked glycan chains

  • Consider testing deglycosylation treatments before Western blot if detection is problematic

  • Fixation methods may differentially preserve these modifications

• Systematic validation approach:

  • Test multiple antibodies across your experimental platforms

  • Include positive controls known to express LYVE1 (e.g., lymphatic vessels in tonsil or liver sinusoids )

  • Validate findings with orthogonal methods (e.g., qPCR for mRNA expression)

  • Consider using genetically modified cells (LYVE1 overexpression or knockout) for definitive validation

• Documentation and reporting:

  • Thoroughly document all experimental conditions that affect detection

  • When publishing, clearly specify antibody clone, catalog number, dilution, and detection method

  • Acknowledge limitations of specific detection platforms in your interpretations

What emerging roles of LYVE1 beyond traditional lymphatic vessel function should researchers consider investigating?

Recent research has uncovered several non-traditional roles for LYVE1 that merit further investigation:

• Neuroimmune interactions in the central nervous system:

  • LYVE1 expression has been detected in human and mouse meninges, co-expressed with PROX1 and MRC1

  • This suggests potential roles in brain fluid dynamics and neuroimmune interactions

  • The presence of LYVE1+ cells in both developing (E12.5-E18) and adult meninges indicates functional significance across the lifespan

  • Future studies should investigate how these LYVE1+ cells in the meninges contribute to CNS homeostasis and disease

• Macrophage-specific LYVE1 functions:

  • LYVE1 expression has been observed in specific macrophage populations, including those differentiated with M-CSF

  • The functional significance of LYVE1 on these cells remains poorly understood

  • Potential roles in hyaluronan clearance, tissue remodeling, or immune regulation warrant investigation

• Role in tumor immunity and immunotherapy response:

  • Peritumoral lymphatic endothelial cells show upregulation of immune regulatory molecules including MHC-II, PD-L1, PD-L2, HVEM, and CD48

  • LYVE1+ vessels may play active roles in tumor immunity beyond passive conduits for metastasis

  • Studies should investigate how targeting LYVE1+ cells might influence immunotherapy efficacy

• Liver-specific functions:

  • The strong expression of LYVE1 in liver sinusoidal endothelial cells suggests specialized functions

  • Potential roles in hyaluronan homeostasis, liver immunity, or response to injury deserve exploration

  • Studies in the context of liver pathologies may reveal novel LYVE1 functions

• Developmental biology applications:

  • The presence of LYVE1 during embryonic development of various tissues suggests roles beyond mature lymphatic function

  • Investigating LYVE1 in stem cell biology, tissue patterning, and organogenesis represents an exciting frontier

• Hyaluronan interaction biology:

  • The masked state of LYVE1's hyaluronan binding site in vivo suggests complex regulation

  • Studies exploring the triggers for unmasking this binding site could reveal novel regulatory mechanisms

  • The functional consequences of LYVE1-hyaluronan binding in different cellular contexts merit investigation

What technological developments might enhance the utility of LYVE1 antibodies in clinical and translational research?

Several emerging technological developments hold promise for enhancing LYVE1 antibody applications in clinical and translational research:

• Advanced imaging technologies:

  • Super-resolution microscopy techniques could reveal nanoscale organization of LYVE1 on cell surfaces

  • Light sheet microscopy enables 3D visualization of entire lymphatic networks while preserving spatial relationships

  • Multi-dimensional Microscopic Molecular Profiling (MMMP) allows iterative staining of tissues to create comprehensive molecular maps

  • These technologies could transform our understanding of LYVE1 distribution in complex tissues

• Single-cell technologies integration:

  • Combining LYVE1 antibodies with single-cell RNA-seq through methods like CITE-seq could correlate protein expression with transcriptional states

  • Spatial transcriptomics techniques could map LYVE1 expression alongside the entire transcriptome within tissue contexts

  • These approaches would provide unprecedented insights into heterogeneity among LYVE1+ cells

• AI-assisted image analysis:

  • Machine learning algorithms could enhance quantification of LYVE1+ structures in complex tissues

  • Automated pattern recognition could identify subtle changes in LYVE1 expression or localization associated with disease states

  • This would enable more objective, reproducible, and high-throughput analysis of LYVE1 expression

• In vivo imaging applications:

  • Development of non-invasive imaging techniques using labeled LYVE1 antibodies or fragments

  • PET, SPECT, or optical imaging approaches could visualize lymphatic vessels in living subjects

  • These techniques could enable longitudinal studies of lymphatic function in disease progression and treatment response

• Therapeutic applications:

  • LYVE1-targeted drug delivery systems could specifically target lymphatic vessels or LYVE1+ macrophages

  • Antibody-drug conjugates directed against LYVE1 might disrupt tumor-associated lymphatic vessels

  • Bispecific antibodies linking LYVE1 with immune checkpoint molecules could modulate immune responses within lymphatic microenvironments

• Standardization efforts:

  • Development of recombinant LYVE1 standards for antibody validation

  • Creation of reference materials and protocols for clinical applications

  • These standardization efforts would enhance reproducibility across laboratories and facilitate clinical translation