Lcn2 Antibody

What cell types express Lcn2 and where should researchers focus their immunostaining studies?

Lcn2 expression has been identified in multiple cell types involved in inflammatory and immune responses. Research indicates that neutrophils, astrocytes, and vascular endothelial cells strongly express Lcn2, particularly under pathological conditions. In brain ischemia models (tMCAo), Lcn2 has been detected in astrocytes whose end-feet encircle blood vessels, neutrophils both within blood vessels and in ischemic brain parenchyma, and on the inner surface of vascular endothelial cells . When designing immunostaining experiments, researchers should include markers for these specific cell types (e.g., GFAP for astrocytes, Ly-6B.2 clone 7/4 for neutrophils, and tomato lectin for blood vessels) to properly identify Lcn2-expressing cells in your tissue of interest.

How does Lcn2 expression change during inflammatory conditions?

Lcn2 levels are significantly elevated across multiple autoimmune disease models, including systemic lupus erythematosus (SLE), collagen-induced arthritis, and serum-induced arthritis . Experimental evidence from mouse models shows that Lcn2 mRNA levels become significantly induced after transient middle cerebral artery occlusion (tMCAo) compared to naive conditions . When designing experiments to study Lcn2 in inflammatory contexts, time-course studies are essential to capture peak expression, which typically occurs within 23 hours after inflammatory insult in acute models . Quantitative analysis methods such as ELISA for protein levels and real-time RT-PCR for mRNA expression provide complementary data to understand the dynamics of Lcn2 upregulation.

What controls should be included when validating Lcn2 antibody specificity?

Proper validation of Lcn2 antibody specificity requires several controls. Experiments using Lcn2 knockout (Lcn2KO) mice tissues provide the gold standard negative control, as demonstrated in multiple studies that show complete absence of signal in these tissues . Immunoprecipitation experiments can further validate specificity by showing that the Lcn2 monoclonal antibody (mAb) can specifically immunoprecipitate both recombinant and endogenous Lcn2 proteins . For Western blot applications, researchers should run parallel samples from wild-type and Lcn2KO mice alongside recombinant Lcn2 protein to confirm band specificity at the expected molecular weight (approximately 37 kDa under reducing conditions) .

How should researchers interpret contradictory findings on Lcn2's role in different inflammatory models?

Lcn2 demonstrates context-dependent roles in inflammatory conditions that may appear contradictory. For instance, in acute skin inflammation models, Lcn2KO mice show approximately 50% reduction in inflammation with notably reduced immune cell infiltration compared to wild-type mice . Conversely, in serum-induced arthritis, Lcn2KO mice develop more severe disease with extensive tissue and bone destruction . These divergent outcomes can be attributed to tissue-specific functions of Lcn2 and differential effects on immune cell populations. When designing experiments to study Lcn2 function, researchers should carefully characterize neutrophil versus macrophage infiltration, as Lcn2KO mice show reduced neutrophil infiltration but increased macrophage migration in arthritis models . Multiple inflammation parameters should be measured, including tissue destruction, cellular infiltration, and inflammatory mediator production to fully understand Lcn2's multifaceted roles.

What are the methodological considerations when using Lcn2 antibodies to study iron sequestration mechanisms?

Lcn2's bacteriostatic properties stem from its ability to sequester bacterial iron siderophores, making iron homeostasis studies crucial to understanding its function . When designing experiments to investigate this mechanism, researchers should include conditions that manipulate iron availability. Studies in Lcn2-deficient mice have confirmed that neutrophils lacking Lcn2 are less effective at inhibiting bacterial growth, especially under iron-limiting conditions . Experimental designs should include bacterial growth assays comparing wild-type and Lcn2KO neutrophils under both iron-replete and iron-depleted conditions. Additionally, researchers should consider examining changes in the microbiome composition, as Lcn2 deficiency has been shown to promote expansion of siderophore-dependent bacterial species, affecting gastrointestinal tract inflammation .

How can researchers effectively use Lcn2 antibodies to investigate its role in infectious disease models?

To investigate Lcn2's role in infectious disease, a multifaceted approach is required. Researchers should utilize Lcn2 antibodies for both neutralization experiments and detection purposes. Neutralization studies have demonstrated that administration of monoclonal antibodies against Lcn2 significantly reduces inflammation in wild-type mice, directly implicating Lcn2 in the inflammatory process . For mechanistic studies, comparing bacterial susceptibility between wild-type and Lcn2KO mice under various iron conditions provides insights into Lcn2's bacteriostatic functions . When designing these experiments, researchers should consider both acute and chronic infection models, as Lcn2's role may differ based on infection duration and pathogen type. Immunohistochemical staining should be performed to track neutrophil infiltration and Lcn2 expression at infection sites, with co-localization studies using markers for immune cells and bacteria.

What are the optimal protocols for detecting Lcn2 expression in tissue sections using immunohistochemistry?

For successful immunohistochemical detection of Lcn2 in tissue sections, several methodological considerations are crucial. Based on published protocols, researchers should perform tissue fixation with 4% paraformaldehyde, followed by cryosectioning for optimal antigen preservation. For multiplex immunofluorescence staining, use Lcn2 antibody (such as AF1857) at a concentration of 5 μg/mL , along with cell-type specific markers to identify Lcn2-expressing cells. For brain tissue, combining Lcn2 antibody (labeled green) with tomato lectin (red, for blood vessels) and GFAP antibody (blue, for astrocytes) enables identification of cell-specific expression patterns . Nuclear counterstaining with DAPI further enhances cell identification. Image acquisition should be performed using confocal microscopy with appropriate filter settings to minimize bleed-through between fluorescence channels. For quantification, analyze at least 5 samples per group, and report the percentage of Lcn2-positive cells by type, as demonstrated in previous studies .

What is the most reliable method for quantifying Lcn2 protein levels in biological samples?

For reliable quantification of Lcn2 protein levels in biological samples, ELISA represents the gold standard method. When analyzing brain tissue, homogenization should be performed in RIPA buffer with protease inhibitors, followed by centrifugation to obtain clear supernatant. For blood samples, serum separation should be performed promptly following collection to prevent ex vivo changes in Lcn2 levels . Research indicates that ELISA can effectively detect changes in Lcn2 concentration in both ipsilateral hemispheres of brain tissue and blood sera following experimental manipulations such as tMCAo or antibody treatment . When reporting results, data should be expressed as concentration (ng/ml) with appropriate statistical analysis comparing experimental groups (n=5-10 per group recommended). For statistical robustness, unpaired t-tests (one-tailed for directional hypotheses, two-tailed for non-directional hypotheses) should be employed when comparing two groups .

How should Western blot protocols be optimized for detecting Lcn2 in different sample types?

Western blot detection of Lcn2 requires optimization based on sample type and experimental context. For mouse lung tissue lysates, loading at 0.2 mg/mL concentration has proven effective . Samples should be prepared under reducing conditions for optimal detection. When probing for Lcn2, researchers should use 5 μg/mL of anti-Lcn2 antibody (such as AF1857) followed by appropriate HRP-conjugated secondary antibody (e.g., 1:50 dilution of anti-goat IgG secondary) . Importantly, Lcn2 typically appears at approximately 37 kDa when using a 12-230 kDa separation system . For immunoprecipitation experiments, incubate increasing concentrations of Lcn2 mAb (0, 0.1, 0.5, and 2.5 μg) bound to magnetic beads with your protein sample, followed by Western blot analysis to confirm specific pull-down of Lcn2 . Always include appropriate positive controls (recombinant Lcn2 protein) and negative controls (samples from Lcn2 knockout animals) to validate specificity.

How can researchers differentiate between free and siderophore-bound forms of Lcn2 in experimental samples?

Differentiating between free and siderophore-bound forms of Lcn2 presents a significant challenge in experimental settings. These forms play distinct roles in Lcn2's bacteriostatic function through iron sequestration . To distinguish between these forms, researchers should employ a combination of analytical approaches. Immunoprecipitation with anti-Lcn2 antibodies followed by mass spectrometry can identify bound siderophores. Additionally, size-exclusion chromatography can separate the different molecular weight complexes prior to Western blot analysis. When interpreting results, researchers should note that Lcn2's bacteriostatic properties are primarily observed under iron-limiting conditions, where it can sequester bacterial siderophores . Comparative analysis between wild-type and Lcn2-deficient models under varying iron conditions provides further insights into the functional relevance of these different forms. Fluorescence-based binding assays using labeled siderophores can also quantify the binding capacity of Lcn2 in experimental samples.

What factors contribute to variability in Lcn2 detection across different experimental models?

Several factors contribute to variability in Lcn2 detection across experimental models. Temporal dynamics of Lcn2 expression show significant changes after inflammatory stimuli, with peak expression typically occurring within 23 hours in acute models . Sample collection timing is therefore critical. The anatomical location of inflammation also influences expression patterns; in brain ischemia models, Lcn2 expression varies between vascular endothelial cells, neutrophils, and astrocytes . Additionally, the inflammatory stimulus type substantially impacts expression levels, with immune-complex mediated inflammation showing distinct patterns from infectious stimuli . Genetic background of experimental animals can also contribute to variability, necessitating proper controls including littermates. Technical factors such as antibody clone selection, detection method sensitivity, and sample processing protocols further contribute to inter-laboratory variation. To minimize variability, researchers should standardize sample collection timing, processing methods, and employ multiple detection techniques (e.g., combining protein detection via ELISA with mRNA quantification via RT-PCR) .

How should researchers interpret differences between mRNA and protein levels of Lcn2 in their experimental systems?

Discrepancies between Lcn2 mRNA and protein levels are commonly observed and require careful interpretation. These differences may reflect several biological processes including post-transcriptional regulation, protein stability differences, or compartmentalization effects. Evidence from tMCAo models shows that while Lcn2 mRNA levels increase significantly after ischemic insult, protein levels may show different patterns in tissue versus serum . When designing experiments, researchers should perform parallel analyses of mRNA (via real-time RT-PCR) and protein (via ELISA or Western blot) from the same experimental groups . For accurate interpretation, consider that antibody-based neutralization of Lcn2 protein may affect observed protein levels without necessarily impacting mRNA expression directly. In studies with Lcn2 mAb treatment, researchers have observed significant decreases in Lcn2 protein concentration in both brain tissue and serum while also noting reduced mRNA expression, suggesting potential feedback mechanisms . Time-course studies tracking both mRNA and protein can help elucidate the relationship between transcription, translation, and protein turnover in your specific experimental system.

How can researchers utilize Lcn2 antibodies to investigate interactions between Lcn2 and its receptors?

Investigating interactions between Lcn2 and its receptors requires sophisticated methodological approaches. Two main receptors have been identified: murine NGALR (discovered through expression cloning in COS-7 cells using a cDNA library from murine FL5.12 cells) and LRP2 (first identified in rats through antibody screening) . To study these interactions, researchers should employ co-immunoprecipitation approaches using anti-Lcn2 antibodies to pull down receptor complexes from cellular lysates, followed by receptor-specific detection methods. Surface plasmon resonance (SPR) or biolayer interferometry using purified components can determine binding kinetics and affinities between Lcn2 and its receptors. For cellular studies, immunofluorescence co-localization experiments using antibodies against both Lcn2 and its receptors can visualize interaction sites within tissues or cells . Proximity ligation assays provide another powerful tool to visualize protein-protein interactions in situ. Functional studies comparing wild-type, receptor knockout, and Lcn2 knockout models help elucidate the physiological relevance of these interactions in various contexts, particularly in iron homeostasis and inflammatory conditions.