CD300LG Antibody, FITC conjugated

What is CD300LG and what are its primary structural characteristics?

CD300LG (CD300 antigen-like family member G), also known as nepmucin or CLM-9, is a novel sialomucin belonging to the type-I membrane protein family. Its structure includes several key domains: a hydrophobic signal peptide, a single V-type Ig domain, a classical mucin-like domain, a transmembrane domain, and an intracellular domain . The protein plays significant roles in lymphocyte trafficking and adhesion, being expressed extensively in various organizational venules and capillary endothelial cells. The protein is recognized as a member of the CD300 family and is encoded by the CD300LG gene, with the corresponding UniprotID of Q6UXG3 according to product information databases .

The structural characteristics of CD300LG are particularly important for understanding its function, as the mucin-like domain associates with L-selectin to mediate lymphocyte rolling, while the Ig domain is involved in adhesion processes independent of LFA-1 or VLA-4 adhesion pathways . These distinct functional domains allow CD300LG to participate in multiple stages of lymphocyte interaction with endothelial cells.

What is the expression pattern of CD300LG across different tissue types?

• High expression: Cardiac tissue, liver, spleen, thymus, lung, kidney, skeletal muscle, salivary gland, thyroid, prostate, tongue, and peripheral lymph nodes

• Low expression: Microvessels of the splenic red pulp and thymic medulla

• Minimal to undetectable expression: Immunologically privileged sites including the brain, testis, uterus, and gut

This differential expression pattern suggests that CD300LG expression is regulated by tissue-specific factors and may correlate with the immunological status of the tissue. Notably, CD300LG expression rapidly decreases in lymph nodes receiving acute inflammatory signals, mediated at least in part by TNF-α, and is down-regulated in tumors and tumor-draining lymph nodes . In contrast, CD300LG is induced in high endothelial venule-like blood vessels in chronically inflamed tissues, such as in pancreatic islets in animal models of non-obese diabetes .

What are the standard applications for CD300LG antibody, FITC conjugated?

• Immunohistochemistry: Both fluorescence and enzyme-based methods for tissue section analysis

• Flow cytometry: For detecting CD300LG expression on cell surfaces

• Confocal microscopy: For visualizing CD300LG distribution in tissues or cultured cells

• Co-localization studies: In combination with other endothelial markers like CD31 and PV-1

The FITC conjugation provides advantages for direct detection without the need for secondary antibodies, offering improved signal-to-noise ratios and enabling multiplex staining approaches when combined with antibodies conjugated to other fluorophores.

What are the optimal protocols for immunohistochemistry using CD300LG antibody?

Based on published research methodologies, the following optimized protocols are recommended for immunohistochemical detection of CD300LG:

For fluorescence immunohistochemistry:

• Prepare frozen tissue sections and fix in methanol

• Block with PBS containing 10% FCS and 20 μg/ml rat IgG

• Incubate with FITC-conjugated anti-CD300LG antibody (the direct conjugation eliminates the need for secondary antibody)

• For co-staining, use antibodies against complementary markers (e.g., CD31, PV-1) conjugated to non-overlapping fluorophores

• Mount with appropriate anti-fade medium

• Visualize using a fluorescence microscope with appropriate filter sets for FITC (excitation ~495 nm, emission ~520 nm)

For enzyme-based immunohistochemistry:

• Fix frozen sections and block with FCS and appropriate IgG

• Incubate with anti-CD300LG antibody

• Treat with 0.3% H2O2 to block endogenous peroxidase activity

• Incubate with horseradish peroxidase-conjugated secondary antibody

• Develop signal with Metal Enhanced DAB substrate

• Counterstain as needed and mount for microscopic analysis

These protocols have been validated in research settings and provide reliable detection of CD300LG in tissue sections while minimizing background and ensuring specificity.

How should researchers optimize storage and handling of CD300LG antibody to maintain activity?

Proper storage and handling of CD300LG antibody, FITC conjugated is crucial for maintaining its immunoreactivity and fluorescence properties. Based on manufacturer recommendations and research protocols, the following guidelines should be implemented:

• Storage temperature: Upon receipt, store at -20°C or -80°C to maintain long-term stability

• Avoid freeze-thaw cycles: Repeated freezing and thawing significantly reduces antibody activity and should be minimized

• Aliquoting: Upon initial thawing, divide the antibody into small single-use aliquots before refreezing

• Light protection: FITC is susceptible to photobleaching, so protect the antibody from light exposure during handling and storage

• Buffer conditions: The antibody is provided in a buffer containing 50% glycerol and 0.01M PBS (pH 7.4) with 0.03% Proclin 300 as a preservative . This formulation helps maintain stability

• Working dilution preparation: When preparing working dilutions, use fresh, high-quality buffer systems and prepare only the volume needed for immediate use

Following these recommendations will help ensure consistent antibody performance across experiments and maximize the usable lifespan of the reagent.

What are the recommended validation steps for CD300LG antibody before experimental use?

Before using CD300LG antibody in critical experiments, researchers should perform several validation steps to confirm specificity and optimal working conditions:

• Positive and negative control tissues: Test the antibody on tissues known to express high levels of CD300LG (e.g., kidney, liver) and those with minimal expression (e.g., brain, testis)

• Titration experiments: Perform a series of dilutions to determine optimal concentration for the specific application

• Blocking experiments: Pre-incubate the antibody with recombinant CD300LG protein to confirm binding specificity

• Western blot validation: If appropriate for the research question, confirm that the antibody recognizes a band of the expected molecular weight (~35-40 kDa for CD300LG)

• Comparative analysis with alternative antibody clones: If available, compare results with other anti-CD300LG antibodies

• RT-PCR correlation: Consider correlating protein detection with mRNA expression using the following primers:

  • Forward: 5′-ACCTCAACCCTTCTGCTGTG-3′

  • Reverse: 5′-ATCACGTCCTCCTTGTCGTC-3′

These validation steps are essential for ensuring experimental rigor and reproducibility in CD300LG research.

How can CD300LG antibody be used to investigate lymphocyte trafficking in inflammatory conditions?

CD300LG plays a crucial role in multiple stages of lymphocyte trafficking, making anti-CD300LG antibodies valuable tools for investigating these processes in inflammatory conditions. Advanced research applications include:

• Dynamic expression analysis: CD300LG expression changes during inflammation, with downregulation in acute inflammation (TNF-α mediated) and upregulation in certain chronic inflammatory conditions . Researchers can use sequential tissue sampling and anti-CD300LG staining to track these changes.

• Co-localization with adhesion molecules: Combined staining of CD300LG with other adhesion molecules (selectins, integrins, etc.) can reveal coordinated expression patterns during inflammation.

• In vivo trafficking studies: FITC-conjugated antibodies can be used to track CD300LG-expressing vessels in relation to infiltrating leukocytes in animal models.

• Mechanistic studies: Since CD300LG mediates adhesion through its Ig domain and rolling through its mucin-like domain, domain-specific blocking experiments using the antibody can distinguish between these functions .

• Therapeutic targeting evaluation: The antibody can be used to assess the effects of potential therapeutic interventions targeting CD300LG-mediated trafficking in inflammatory models.

These applications provide insights into the complex interplay between endothelial cells and immune cells during inflammation, potentially leading to novel therapeutic approaches for inflammatory diseases.

What role does CD300LG play in tumor immunology and how can the antibody be used in cancer research?

CD300LG has emerging significance in tumor immunology, with its expression patterns potentially influencing anti-tumor immune responses. Research has revealed several important aspects:

• Downregulation in tumor microenvironments: CD300LG expression is reduced in tumors and tumor-draining lymph nodes, which may contribute to immune evasion by limiting lymphocyte trafficking to the tumor site .

• Impact on cytotoxic cell function: Studies have shown that CD300LG induction can significantly improve the killing activity of cytokine-induced killer (CIK) cells against various target cells, including tumor cells .

• Dual role in tumor immunity: CD300LG can enhance immune cell activity whether expressed on effector cells or target cells, suggesting complex immunomodulatory properties .

Researchers can use CD300LG antibodies in cancer research through several approaches:

• Tumor vasculature characterization: Assess CD300LG expression patterns in tumor-associated blood vessels compared to normal tissue vasculature

• Immune infiltrate correlation studies: Examine relationships between CD300LG expression and tumor-infiltrating lymphocyte densities

• Therapeutic modification assessment: Monitor changes in CD300LG expression following immune checkpoint inhibition or other immunotherapies

• Prognostic biomarker evaluation: Investigate correlations between CD300LG expression patterns and clinical outcomes

These applications may provide new insights into mechanisms of tumor immune evasion and potential strategies for enhancing anti-tumor immunity.

What techniques can be used to study CD300LG's influence on cytotoxic immune cell activity?

Recent research has demonstrated that CD300LG can significantly enhance the cytotoxic activity of immune cells, suggesting important immunomodulatory functions. Researchers can investigate these effects using several sophisticated approaches:

• Transfection-based systems: Create CD300LG-expressing cell lines (as done with hCD300LG-γ/pEGFP-C3 constructs) to study the effects on immune cell interactions

• CIK cell induction protocols: Prepare cytokine-induced killer cells using different induction methods, including CD300LG-containing cell lysates as described in the literature. The basic protocol involves:

  • Isolating human peripheral blood mononuclear cells

  • Inducing with cell lysates from CD300LG-expressing cells

  • Concurrent treatment with standard CIK inductive agents

  • Measuring cytotoxicity against various target cells

• Cytotoxicity assays: Compare killing activities of CD300LG-induced vs. standard CIK cells against multiple target cell types using methods such as:

  • Chromium release assays

  • Flow cytometry-based cytotoxicity assays

  • Real-time cell analysis systems

• Blocking experiments: Use anti-CD300LG antibodies to block specific domains and determine which structural elements are essential for enhanced cytotoxicity

• Mechanism analysis: Investigate the molecular pathways through which CD300LG enhances cytotoxic activity, potentially involving:

  • Adhesion molecule upregulation

  • Cytokine production profiles

  • Cytotoxic granule release kinetics

The significant enhancement of CIK cell activity following CD300LG induction suggests potential applications in cancer immunotherapy development .

What are common technical challenges when working with CD300LG antibody and how can they be addressed?

Researchers working with CD300LG antibody may encounter several technical challenges. Here are common issues and their solutions:

• Variable staining intensity across tissues:

  • Challenge: CD300LG expression varies significantly between tissue types and vascular beds

  • Solution: Include known positive control tissues in each experiment and optimize antibody concentration for each specific tissue type

• False negative results in tissues with low expression:

  • Challenge: Immunologically privileged sites have minimal CD300LG expression

  • Solution: Use signal amplification methods (tyramide signal amplification or photomultiplier enhancement) for tissues with low expression levels

• FITC photobleaching:

  • Challenge: FITC is susceptible to photobleaching during extended imaging sessions

  • Solution: Use anti-fade mounting media, minimize exposure time, and consider sequential rather than simultaneous image acquisition

• Cross-reactivity concerns:

  • Challenge: CD300LG belongs to a family of related proteins with structural similarities

  • Solution: Validate specificity using knockout/knockdown controls or competitive binding assays with recombinant proteins

• Fixation-sensitive epitopes:

  • Challenge: Some epitopes may be altered by certain fixation methods

  • Solution: Compare methanol fixation (recommended in protocols) with paraformaldehyde or other fixatives to determine optimal preservation of the target epitope

Addressing these challenges through methodological refinements will enhance the reliability and reproducibility of CD300LG research.

How should researchers design multiplex immunofluorescence experiments involving CD300LG antibody?

Multiplex immunofluorescence studies involving CD300LG antibody require careful planning to achieve optimal results. The following design considerations are recommended:

• Compatible fluorophore selection:

  • The FITC conjugate of CD300LG antibody has excitation/emission peaks around 495/520 nm

  • Select additional fluorophores with minimal spectral overlap, such as:

    • Cy3 (~550/570 nm) for secondary markers

    • Alexa Fluor 647 (~650/668 nm) for tertiary markers

    • DAPI (~358/461 nm) for nuclear counterstaining

• Panel design strategies:

  • For vascular studies, combine with endothelial markers like CD31 and PV-1

  • For leukocyte trafficking studies, include markers for specific immune cell populations

  • For inflammatory models, consider adding markers for activation states

• Sequential staining protocol:

  1. Apply FITC-conjugated CD300LG antibody first

  2. Block with appropriate IgG to prevent cross-reactivity

  3. Apply additional primary antibodies with compatible host species

  4. Use secondary antibodies with minimal cross-reactivity

  5. Include proper controls for each antibody individually

• Image acquisition considerations:

  • Capture each fluorophore channel separately using appropriate filter sets

  • Adjust exposure times to accommodate different expression levels

  • Consider spectral unmixing for closely overlapping fluorophores

• Analysis approaches:

  • Use colocalization analysis software to quantify marker associations

  • Consider automated vessel segmentation for morphometric analysis

  • Implement machine learning approaches for complex pattern recognition

These strategies will enable researchers to obtain comprehensive data on CD300LG expression and function in relation to other molecular markers.

What are emerging research areas regarding CD300LG's role in immune regulation?

Several promising research directions are emerging regarding CD300LG's role in immune regulation:

• Chronic inflammation modulation: CD300LG is induced in high endothelial venule-like blood vessels in chronically inflamed tissues, such as pancreatic islets in animal models of non-obese diabetes . This presents opportunities to investigate its role in autoimmune diseases and potential therapeutic targeting.

• Dynamic regulation mechanisms: The rapid changes in CD300LG expression in response to local inflammatory signals (particularly TNF-α-mediated downregulation) suggest sophisticated regulatory mechanisms that warrant further exploration .

• Tumor immunity: The observation that CD300LG expression is reduced in tumors and tumor-draining lymph nodes points to potential involvement in tumor immune evasion mechanisms . The finding that CD300LG induction enhances CIK cell cytotoxicity further suggests therapeutic applications in cancer immunotherapy .

• Lymphocyte subset interactions: Research exploring whether CD300LG differentially affects various lymphocyte subsets (CD4+ T cells, CD8+ T cells, B cells, NK cells) could reveal subset-specific regulatory mechanisms.

• Ligand identification and characterization: Further work is needed to fully characterize the ligand(s) for CD300LG expressed on activated T cells, as suggested by findings in inflamed pancreatic tissue .

These emerging areas represent fertile ground for innovative research with potential clinical applications in inflammatory diseases, autoimmunity, and cancer.

How might CD300LG antibodies contribute to developing new immunotherapeutic approaches?

Based on current research findings, CD300LG antibodies may contribute to novel immunotherapeutic approaches in several ways:

• Enhanced CIK cell therapy: CD300LG induction significantly improves the killing activity of CIK cells against various target cells, including tumor cells . This suggests potential for:

  • Development of CD300LG-induced CIK cells as an improved cellular therapy

  • Creation of engineering protocols to express CD300LG on therapeutic immune cells

  • Combination approaches with established adoptive cell therapies

• Targeted vascular modulation: Given CD300LG's differential expression in various tissues and its downregulation in tumors , antibody-based approaches could:

  • Reverse immune suppression in tumor microenvironments by targeting CD300LG regulation

  • Enhance lymphocyte trafficking to tumors by manipulating CD300LG expression

  • Reduce inappropriate inflammation by modulating CD300LG in specific vascular beds

• Diagnostic applications: CD300LG antibodies might serve as:

  • Biomarkers for assessing vascular inflammation

  • Indicators of immunologically active vs. suppressed tumor microenvironments

  • Tools for monitoring response to immunotherapies

• Combination therapy development: Research into CD300LG's interactions with established immunotherapy targets (checkpoint molecules, cytokines) could identify synergistic combination approaches.

These potential applications represent promising directions for translational research, potentially expanding the repertoire of immunotherapeutic approaches for inflammatory diseases and cancer.