LYVE1 Antibody

What is LYVE1 and why is it important in lymphatic research?

LYVE1 (Lymphatic Vessel Endothelial Hyaluronan Receptor 1) is a 322-amino acid type-I integral membrane glycoprotein primarily expressed on lymphatic endothelial cells (LECs) and some populations of macrophages. It functions as a major receptor for hyaluronan (HA), a large mucopolysaccharide. The extracellular domain of LYVE1 contains a conserved hyaluronan binding domain also found in CD44 . LYVE1 is a characteristic marker protein for lymphatic endothelial cells, along with podoplanin, PROX-1, Tie-2, and VEGFR-3 . Its importance in research stems from its role in lymphatic hyaluronan transport and potential involvement in tumor metastasis . The lymphatic system is crucial for immune surveillance, as it transports proteins and cells (especially dendritic cells) throughout the body .

What are the differences between various LYVE1 antibody types available for research?

LYVE1 antibodies are available in both monoclonal and polyclonal formats, each with specific advantages for different applications. Monoclonal antibodies like ALY7 offer high specificity and consistency between batches, making them ideal for standardized protocols . Polyclonal antibodies, such as 28321-1-AP, recognize multiple epitopes and can provide stronger signals in some applications . Researchers should consider species reactivity (human, mouse, rat) based on their experimental model . Antibodies may be unconjugated or directly conjugated to fluorophores like AlexaFluor for direct visualization . When selecting an antibody, consider the validation data for your specific application (WB, IHC, IF, ELISA) and host species compatibility with your secondary detection system .

How does LYVE1 antibody staining compare to other lymphatic vessel markers?

LYVE1 serves as one of several complementary lymphatic markers, each with distinct expression patterns and advantages. Unlike pan-endothelial markers that label both blood and lymphatic vessels, LYVE1 provides specificity for lymphatic endothelium . LYVE1 antibodies typically yield strong staining of lymphatic vessel walls and valves with minimal background in properly optimized protocols . In comparative studies, LYVE1 shows minimal to no staining of blood vessels, confirming its specificity as a lymphatic marker . For comprehensive lymphatic characterization, researchers often employ LYVE1 alongside other markers such as podoplanin, PROX-1, Tie-2, and VEGFR-3 . When analyzing complex tissues, particularly in tumors, co-staining with multiple lymphatic markers may be necessary to differentiate lymphatic structures from LYVE1-positive macrophages .

What are the optimal conditions for using LYVE1 antibodies in immunohistochemistry (IHC)?

For optimal IHC results with LYVE1 antibodies, tissue preparation and antigen retrieval are critical factors. For frozen sections, antibodies like ALY7 can be used at a concentration of 2.5 μg/mL for immunofluorescent microscopy . For formalin-fixed, paraffin-embedded tissues, antigen retrieval is essential - using either TE buffer (pH 9.0) or citrate buffer (pH 6.0) depending on the antibody . The recommended dilution range for IHC applications is typically 1:500-1:2000 for polyclonal antibodies like 28321-1-AP . Antibody titration is strongly recommended to determine optimal concentration for each specific tissue type and fixation method. When staining lymphatic vessels, include positive control tissues such as lymph node or intestine where lymphatics are abundant and clearly identifiable . For visualization, both chromogenic and fluorescent detection methods are compatible with LYVE1 antibodies, with fluorescence offering advantages for co-localization studies .

How should researchers approach Western blot analysis using LYVE1 antibodies?

When performing Western blot analysis with LYVE1 antibodies, researchers should anticipate detecting bands between 50-70 kDa, despite the calculated molecular weight of 35 kDa . This discrepancy is due to extensive glycosylation and post-translational modifications of the LYVE1 protein . Sample preparation is critical - protein lysates from LYVE1-rich tissues such as lymph nodes, fetal lung, and fetal spleen yield stronger signals than tissues with minimal lymphatic presence like brain . For optimal results with polyclonal antibodies like 28321-1-AP, use dilutions between 1:1000-1:4000 . When interpreting Western blot results, be aware that multiple bands may be observed, representing both full-length LYVE1 and soluble fragments as described in the literature . For immunoprecipitation applications, use approximately 1/20 dilution of antibody for pull-down followed by 1/1000 dilution for detection blotting . Include positive controls such as human fetal lung or lymph node lysates which express significant amounts of LYVE1 protein .

What methods provide the best results for flow cytometric analysis of LYVE1?

For successful flow cytometric analysis of LYVE1, sample preparation and antibody concentration are crucial factors. When using monoclonal antibodies like ALY7, the recommended concentration is ≤0.125 μg per million cells in a 100 μL total staining volume . Cell isolation techniques should preserve surface epitopes - consider using enzyme-free dissociation buffers when preparing samples from tissues expressing LYVE1. For analyzing primary lymphatic endothelial cells, include additional markers (e.g., CD31+/podoplanin+) to define the lymphatic endothelial population accurately. When analyzing transfected cell lines expressing LYVE1, include both negative (untransfected) and positive controls to establish gating strategies. Titration experiments are essential, as both under and over-staining can affect result interpretation. For multicolor flow cytometry, consider fluorophore brightness and compensation requirements when selecting conjugated LYVE1 antibodies. When analyzing LYVE1 expression on macrophage subsets, co-staining with macrophage markers (e.g., F4/80, CD11b) is necessary for accurate population identification.

How can fluorescent LYVE1 antibodies be used for in vivo lymphatic vessel imaging?

Fluorescent LYVE1 antibodies enable powerful real-time visualization of lymphatic vessels and tracking of cell trafficking in vivo. Conjugation of LYVE1 antibodies with fluorophores like AlexaFluor creates a specific and durable signal for visualizing lymphatic architecture . For optimal results, inject approximately 2.4 μg of conjugated LYVE1 antibody in a 50 μL volume into tissues surrounding the area of interest (e.g., inguinal lymph node) . The fluorescent signal peaks around 4 hours post-injection and remains detectable for at least 48 hours, offering a substantial window for repeated imaging sessions . This approach is significantly superior to traditional lymphangiography using FITC-dextran or control IgG, which show minimal signal at 4 hours and no detectable signal by 12 hours post-injection . For tumor cell trafficking studies, this technique can be combined with cells expressing red fluorescent protein (RFP), enabling color-coded tracking of tumor cells within green fluorescent-labeled lymphatic vessels . Specificity verification through ex vivo analysis confirms that the signal localizes to lymphatic endothelium, with clear delineation of vessel walls and valves and minimal background staining .

What considerations are important when designing lymphangiogenesis studies using LYVE1 antibodies?

When designing lymphangiogenesis studies, researchers must carefully consider several methodological aspects to ensure reliable quantification and interpretation. Selection of appropriate tissue sections is crucial - standardize the anatomical regions being analyzed to minimize variability in lymphatic vessel density. Implement consistent imaging parameters including magnification, exposure time, and threshold settings across all experimental and control samples. For accurate quantification, establish clear criteria for identifying positive LYVE1 staining versus background or non-specific signals. In tumor models, be aware that LYVE1 can be expressed by a subset of tumor-associated macrophages, necessitating double-staining with macrophage markers for accurate vessel identification . When analyzing lymphatic vessel density, consider multiple parameters including vessel number, size, area, and perimeter rather than relying on a single metric. For intervention studies (anti-lymphangiogenic therapies), establish appropriate baseline measurements and time points for analysis that align with the expected biological response. Include relevant positive and negative controls for antibody specificity, and consider using alternative lymphatic markers (podoplanin, PROX-1) in parallel for validation.

How can researchers distinguish between LYVE1 expression on lymphatic vessels versus macrophages?

Distinguishing LYVE1 expression on lymphatic endothelial cells versus macrophages requires careful experimental design and multiple validation approaches. Co-immunostaining is essential - combine LYVE1 with endothelial markers (CD31, VEGFR-3) and macrophage markers (F4/80, CD11b, CD68) to differentiate cell populations . Morphological assessment provides additional discrimination - lymphatic vessels typically show distinct structures with clear lumens and valve formations, whereas macrophages appear as individual cells with irregular shapes . Functional uptake studies can further distinguish cell types - in vivo administration of fluorescent-labeled LYVE1 antibodies primarily labels functional lymphatic vessels since uptake depends on lymphatic flow, with minimal contribution from tissue macrophages . For challenging tissues such as tumors or inflamed tissue, consider using lineage-specific transcription factors like PROX-1 (specific for lymphatic endothelium) in combination with LYVE1. Flow cytometric analysis can separate these populations based on differential expression levels and co-expression patterns of multiple markers. Remember that while LYVE1 expression has been reported on some macrophage subsets, the likelihood of significant contribution to in vivo imaging signals is low due to the functional nature of antibody uptake in lymphatic vessels .

Why does LYVE1 show variability in molecular weight across different Western blot studies?

The observed variability in LYVE1 molecular weight on Western blots stems from several biological and technical factors that researchers should consider when interpreting results. While the calculated molecular weight of LYVE1 is approximately 35 kDa, it typically appears as bands between 50-70 kDa due to extensive post-translational modifications, particularly glycosylation . This glycosylation pattern can vary between tissue types and physiological states, contributing to band pattern differences between samples. Additionally, researchers should be aware that LYVE1 exists in both full-length and soluble fragments, resulting in multiple bands on Western blots . The soluble forms result from proteolytic cleavage of the membrane-bound receptor and have been documented in the literature (PMID: 29262593, 26966180, 26926389) . Sample preparation methods, particularly the lysis buffer composition and protease inhibitors used, can affect the band pattern observed. The specific epitope recognized by different antibody clones may also contribute to variability, as some antibodies may preferentially detect certain forms or modified versions of the protein. When comparing results between studies, consider both the antibody clone used and the sample origin, as human, mouse, and rat LYVE1 may show slight differences in molecular weight and band patterns .

What strategies help optimize signal-to-noise ratio when using LYVE1 antibodies in immunostaining?

Achieving optimal signal-to-noise ratio with LYVE1 antibodies requires careful attention to several experimental parameters. Begin with proper antibody titration - testing a range of concentrations is essential, as both under and over-staining can compromise result quality. For ALY7 monoclonal antibody, start with recommended concentrations (2.5 μg/mL for immunofluorescence) and adjust based on your specific tissue and detection system . Implement rigorous blocking protocols using appropriate blocking agents (e.g., 5% normal serum, BSA, or commercial blocking solutions) to minimize non-specific binding. Consider tissue fixation methods carefully - overfixation can mask epitopes while inadequate fixation may compromise tissue morphology; optimize fixation time and fixative concentration for your specific tissue. For paraffin-embedded tissues, evaluate different antigen retrieval methods as LYVE1 epitopes may respond differently to citrate buffer (pH 6.0) versus TE buffer (pH 9.0) . Include properly matched isotype controls at the same concentration as the primary antibody to distinguish specific from non-specific staining . For tissues with high autofluorescence (liver, brain), consider additional treatments such as Sudan Black B or commercial autofluorescence quenching reagents before applying antibodies. When performing multi-color immunofluorescence, select fluorophores with minimal spectral overlap and include single-stained controls for accurate compensation settings.

How should researchers address contradictory LYVE1 expression data in different experimental systems?

When confronting contradictory LYVE1 expression data across experimental systems, a systematic troubleshooting approach is essential for resolving discrepancies. Start by comparing antibody clones - different antibodies may recognize distinct epitopes, potentially explaining variable staining patterns . Verify antibody specificity through multiple methods, including positive/negative control tissues, knockdown validation, and comparison with alternative LYVE1 antibodies. Assess the impact of sample preparation methods as fixation protocols, antigen retrieval techniques, and tissue processing can significantly affect epitope accessibility and antibody binding. Consider the physiological state of the tissue being analyzed - inflammation, hypoxia, or disease states may alter LYVE1 expression or glycosylation patterns. Compare detection methods systematically - discrepancies might arise when comparing results obtained through different techniques (IHC vs. IF vs. flow cytometry vs. Western blot). Re-evaluate data interpretation frameworks, particularly for distinguishing lymphatic vessels from LYVE1-positive macrophages in complex tissues. Verify that the observed structures are indeed lymphatic vessels by confirming co-expression with other lymphatic markers (podoplanin, PROX-1) or functional characteristics . For cross-species comparisons, be aware that human, mouse, and rat LYVE1 may show subtle differences in expression patterns, antibody reactivity, or molecular weight . When all else fails, consider biological variability - different tissue microenvironments may genuinely exhibit different LYVE1 expression patterns.

How are LYVE1 antibodies being utilized in tumor microenvironment and metastasis research?

LYVE1 antibodies have become instrumental tools for investigating the complex relationships between lymphatic vessels and cancer progression. Researchers are employing fluorescent-labeled LYVE1 antibodies to track real-time tumor cell migration through lymphatic vessels, offering unprecedented insights into the mechanisms of lymphatic metastasis . This approach enables simultaneous visualization of both the lymphatic architecture (labeled with green fluorescent LYVE1 antibodies) and tumor cells (expressing red fluorescent protein), allowing direct observation of tumor-lymphatic interactions . Beyond visualization, LYVE1 antibodies are being used to quantify peritumoral and intratumoral lymphangiogenesis, a process strongly associated with lymph node metastasis in many cancer types. The dual expression of LYVE1 on both lymphatic vessels and specific macrophage populations makes it particularly valuable for studying the tumor immune microenvironment, as these cell types may have different roles in tumor progression. LYVE1 antibodies are also being employed to investigate the role of tumor-associated macrophages in promoting lymphangiogenesis through LYVE1-dependent mechanisms. For therapeutic development, these antibodies serve as screening tools for potential anti-lymphangiogenic compounds that might inhibit metastatic spread. When designing such studies, researchers should carefully distinguish between LYVE1-positive lymphatic vessels and macrophages using multi-parameter analysis and morphological assessment .

What recent advances have been made in understanding LYVE1's molecular interactions and signaling?

Recent research using LYVE1 antibodies has expanded our understanding of this receptor beyond its traditional role as a lymphatic marker. Investigation of LYVE1's binding characteristics has revealed that its interaction with hyaluronan in vivo is regulated by sialylated O-linked glycan chains that mask the binding site, suggesting that binding requires specific modification or unmasking events to expose the active site . This regulated binding may play important roles in controlling lymphatic uptake and transport of hyaluronan and associated molecules. Studies have demonstrated that although LYVE1 can bind hyaluronan in vitro, the physiological regulation is more complex, potentially involving tissue-specific or condition-specific modifications . Advanced structural analyses using epitope-specific antibodies are helping to map the functional domains of LYVE1 and their interactions with ligands beyond hyaluronan. Research into LYVE1's role in macrophages has revealed potential functions in immune regulation and tissue homeostasis distinct from its role in lymphatic vessels. LYVE1 antibodies are being used to investigate potential signaling pathways downstream of receptor activation, which may influence lymphatic endothelial cell behavior including migration, tube formation, and valve development. The discovery that hematopoietic stem cells can differentiate into LYVE1-positive lymphatic endothelial cells that integrate into the endothelium in normal and metastatic tissue opens new avenues for understanding lymphatic vessel development and remodeling .

What methodological advances are improving the specificity and sensitivity of LYVE1 detection?

Technological innovations are continuously enhancing the utility of LYVE1 antibodies for increasingly sophisticated research applications. Development of highly specific monoclonal antibodies like EPR21857 and ALY7 has improved the reliability of LYVE1 detection across multiple applications . Fluorophore conjugation technologies now enable direct labeling of LYVE1 antibodies with bright, photostable fluorophores that provide superior signal duration compared to traditional lymphangiography methods using FITC-dextran . Advanced microscopy techniques including super-resolution imaging and intravital microscopy are being combined with fluorescent LYVE1 antibodies to visualize lymphatic vessel structure and function at unprecedented resolution. Multiplexed immunostaining approaches allow simultaneous detection of LYVE1 alongside numerous other markers, enabling comprehensive characterization of the lymphatic microenvironment. The application of tissue clearing techniques is expanding three-dimensional visualization of LYVE1-positive structures throughout intact organs and tissues. Single-cell analysis methods incorporating LYVE1 antibodies are revealing previously unrecognized heterogeneity within lymphatic endothelial cell populations. For Western blot applications, improved detection systems have enhanced sensitivity for detecting both full-length LYVE1 and its soluble fragments . In flow cytometry, optimized multicolor panels incorporating LYVE1 enable more precise identification and isolation of lymphatic endothelial cells from complex tissues. These technological advances collectively provide researchers with more powerful and precise tools for investigating LYVE1 biology in both physiological and pathological contexts.

Table 1: Recommended LYVE1 Antibody Applications and Conditions