LRRK2 Antibody, FITC conjugated

What is LRRK2 and why is it significant for Parkinson's disease research?

LRRK2 (Leucine-rich repeat kinase 2) is a multifunctional serine/threonine-protein kinase that phosphorylates a broad range of proteins involved in neuronal plasticity, innate immunity, autophagy, and vesicle trafficking . It functions as a key regulator of RAB GTPases by controlling the GTP/GDP exchange and interaction partners of RABs through phosphorylation . LRRK2 is particularly significant for Parkinson's disease research because it represents one of the most commonly mutated genes in familial Parkinson's disease . Mutations in LRRK2, particularly in its kinase domain, are strongly associated with the pathogenesis of both familial and sporadic forms of Parkinson's disease . The G2019S mutation, for example, is a well-characterized pathogenic variant that increases kinase activity . Understanding LRRK2 function and dysregulation provides critical insights into disease mechanisms and potential therapeutic targets, as orally bioavailable, brain-penetrant LRRK2 kinase inhibitors are now in later stages of clinical development .

What are the key applications for FITC-conjugated LRRK2 antibodies in research?

FITC-conjugated LRRK2 antibodies provide several advantages for research applications that require direct fluorescent detection without secondary antibody steps. The primary applications include:

• Flow cytometry analysis of LRRK2 expression in immune cells, particularly in peripheral blood mononuclear cells (PBMCs) and neutrophils .

• Immunofluorescence microscopy for direct visualization of LRRK2 localization within cells and tissues, especially useful in co-localization studies with other proteins like α-synuclein .

• ELISA-based applications for detecting LRRK2 in biological samples, as specified in the antibody properties .

• Monitoring changes in LRRK2 expression levels in response to stimuli or treatments in live cells .

The FITC conjugation enables direct detection of the antibody-antigen complex without requiring additional staining steps, simplifying protocols and reducing background when examining LRRK2 expression patterns or interactions with other cellular components .

How should researchers validate the specificity of LRRK2 antibodies in their experimental systems?

Validating antibody specificity is crucial for reliable LRRK2 research. Researchers should implement the following methodological approaches:

• Positive and negative controls: Use samples with confirmed LRRK2 expression (e.g., neutrophils) as positive controls and LRRK2 knockout/knockdown samples as negative controls .

• LRRK2 inhibitor treatment: Treat cells with selective LRRK2 inhibitors like MLi-2 (100 nM) to confirm that observed phosphorylation events are LRRK2-dependent . This approach is particularly useful for validating phospho-specific antibodies recognizing LRRK2 substrates like Rab10.

• Western blot analysis: Confirm antibody specificity by detecting the expected molecular weight bands of LRRK2 (full-length at ~286 kDa and potential truncated forms at ~170 kDa) . Note that LRRK2 may present multiple species that should be carefully characterized.

• Cross-reactivity assessment: Test against closely related proteins like LRRK1 to ensure specificity, as these proteins share structural similarities but have distinct functions and disease associations .

• Epitope mapping: Verify that the antibody recognizes the intended region of LRRK2, particularly important when distinguishing between full-length protein and truncated forms .

The Michael J. Fox Foundation has contributed significantly to resolving previous issues with LRRK2 antibody specificity by developing well-characterized antibodies, which should be considered as reference standards when validating new antibodies .

What are the optimal sample preparation techniques for LRRK2 detection in different cell types?

Optimal sample preparation for LRRK2 detection varies by cell type and application:

For peripheral blood neutrophils:

• Isolate neutrophils using immunomagnetic negative isolation to achieve >97% purity .

• Confirm cell purity using flow cytometry with neutrophil markers like CD66b-FITC .

• Lyse cells in buffer containing diisopropyl fluorophosphate (DIFP) to suppress intrinsic serine protease activity that is high in these cells .

• Process samples quickly at 4°C to prevent protein degradation and dephosphorylation .

For peripheral blood mononuclear cells (PBMCs):

• Consider the heterogeneity of PBMCs, as only a subset (monocytes and contaminating neutrophils) express significant levels of LRRK2 .

• For monocyte activation experiments, treat isolated cells with IFN-γ to increase LRRK2 expression before analysis .

• Use fresh samples when possible, as LRRK2 may degrade during extended storage .

For neuronal samples:

• For brain tissue samples, rapid extraction and processing are essential to maintain protein integrity .

• For co-localization studies with α-synuclein, formaldehyde fixation followed by permeabilization is often used .

• When preparing brain lysates, use protease and phosphatase inhibitors to preserve both LRRK2 and its post-translational modifications .

What are the recommended storage and handling conditions for FITC-conjugated LRRK2 antibodies?

To maintain optimal antibody performance, follow these storage and handling recommendations:

• Upon receipt, store FITC-conjugated LRRK2 antibodies at -20°C or -80°C as specified by the manufacturer .

• Avoid repeated freeze-thaw cycles as they can damage the FITC fluorophore and reduce antibody activity .

• When using the antibody, aliquot working volumes to minimize freeze-thaw cycles of the stock solution.

• Store in buffer containing 50% glycerol and 0.01M PBS (pH 7.4) with appropriate preservative (e.g., 0.03% Proclin 300) to maintain stability .

• Protect from light during all handling steps to prevent photobleaching of the FITC fluorophore.

• For long-term storage of FITC-conjugated antibodies, -80°C is preferable to -20°C for maintaining fluorescence intensity.

• Follow manufacturer's recommendations for specific dilution factors based on application (e.g., ELISA, flow cytometry) .

How can researchers quantify LRRK2 kinase pathway activity in primary human samples?

Quantifying LRRK2 kinase pathway activity in primary human samples requires precise methodology focusing on its physiological substrates. A robust approach involves:

• Measuring Rab10 phosphorylation: LRRK2-mediated phosphorylation of Rab10 at Thr73 serves as a reliable readout of LRRK2 kinase activity . This can be quantified using the selective MJFF-pRab10 monoclonal antibody specifically recognizing this phospho-epitope.

• Using peripheral blood neutrophils: These cells are ideal for monitoring LRRK2 activity because they:

  • Express relatively high levels of both LRRK2 and its substrate Rab10

  • Are abundant and homogeneous

  • Can be easily isolated from blood samples with >97% purity

  • Yield sufficient protein (0.2-1.4 mg) from 20 ml of blood for multiple assays

• Employing quantitative immunoblotting: The recommended protocol involves:

  • Treating samples with/without selective LRRK2 inhibitor (e.g., 100 nM MLi-2) to establish baseline

  • Lysing cells in buffers containing protease inhibitors like DIFP

  • Analyzing both LRRK2 Ser935 phosphorylation (upstream marker) and Rab10 Thr73 phosphorylation (direct substrate)

• Normalization approach: Calculate the ratio of phosphorylated Rab10 to total Rab10 protein to account for expression differences between samples .

This method provides a pharmacodynamic readout that could be valuable for LRRK2 inhibitor trials, patient stratification, and monitoring disease progression .

What experimental approaches can detect interactions between LRRK2 and α-synuclein in Parkinson's disease models?

Investigating LRRK2 and α-synuclein interactions requires multiple complementary approaches:

• Co-immunoprecipitation (Co-IP): The gold standard for assessing direct protein interactions.

  • Use well-characterized LRRK2 antibodies (like those from the Michael J. Fox Foundation)

  • Perform reciprocal Co-IPs (pulling down with anti-LRRK2 and probing for α-synuclein, and vice versa)

  • Include appropriate controls (IgG control, LRRK2 knockout samples)

• Immunofluorescence co-localization:

  • Apply dual labeling with FITC-conjugated LRRK2 antibody and differently labeled α-synuclein antibody

  • Analyze co-localization in neuronal cells or brain sections, particularly in Lewy bodies

  • Quantify co-localization using digital image analysis software

• Proximity ligation assay (PLA):

  • More sensitive than conventional co-localization for detecting protein-protein interactions

  • Provides signal only when proteins are within 40 nm of each other

  • Allows quantification of interaction frequency in different cell compartments

• Cell models of α-synuclein inclusion formation:

  • Study whether LRRK2 co-localizes with α-synuclein in inclusion bodies

  • Investigate if LRRK2 mutations affect this co-localization

  • Determine whether LRRK2 kinase inhibitors modify the interaction

• Biochemical correlation studies:

  • Assess whether increased LRRK2 protein levels correlate with increased phosphorylated α-synuclein in affected brain regions

  • Compare LRRK2 and α-synuclein levels in different brain regions and disease stages

These approaches have revealed that LRRK2 protein levels increase in association with rising levels of phosphorylated α-synuclein in Parkinson's disease brain regions, suggesting a functional relationship between these proteins .

How can researchers distinguish between full-length LRRK2 and truncated forms in experimental samples?

Distinguishing between LRRK2 protein variants requires careful technical consideration:

• Domain-specific antibodies: Use antibodies targeting different regions:

  • C-terminal antibodies detect both full-length (~286 kDa) and N-terminally truncated (~170 kDa) forms

  • N-terminal antibodies specifically detect only full-length LRRK2

  • Compare results from both antibody types to identify truncated species

• SDS-PAGE optimization:

  • Use low percentage (6-8%) polyacrylamide gels for better separation of high molecular weight proteins

  • Employ gradient gels (4-12%) to simultaneously resolve both large and small LRRK2 species

  • Extend running time to achieve clear separation between the 286 kDa and 170 kDa bands

• Quantification methodology:

  LRRK2 Species	Molecular Weight	Detection Method	Relative Abundance in Neutrophils
  Full-length	~286 kDa	N & C-terminal antibodies	1× (reference)
  N-terminally truncated	~170 kDa	C-terminal antibodies only	2-3× full-length

• Phosphorylation analysis:

  • Both full-length and truncated forms can be phosphorylated at Ser935

  • Treatment with LRRK2 inhibitors (e.g., MLi-2) causes dephosphorylation of both forms

  • The truncated form may show greater sensitivity to inhibitor-induced dephosphorylation

• Functional differences:

  • Monitor changes in relative abundance under different conditions

  • Note that MLi-2 treatment can reduce the amount of the ~170 kDa species without affecting full-length levels

  • Investigate whether truncated forms have distinct localization or interaction patterns

Careful characterization of these LRRK2 species is essential for accurate interpretation of experimental results, particularly when assessing the effects of potential therapeutics.

What protocols can effectively measure LRRK2 expression changes in immune cells following activation stimuli?

LRRK2 expression in immune cells is dynamically regulated during activation. To effectively measure these changes:

• Isolation of specific immune cell populations:

  • For monocytes/macrophages: Use negative selection immunomagnetic separation

  • For neutrophils: Apply immunomagnetic negative isolation achieving >97% purity

  • Confirm purity by flow cytometry using appropriate markers (CD14, CD16 for monocytes; CD66b for neutrophils)

• Stimulation protocols:

  • For monocyte activation: Treat with IFN-γ (most potent LRRK2 inducer), GM-CSF, M-CSF, or LPS

  • Culture cells at 37°C in 5% CO₂ humidified atmosphere

  • Refresh media containing stimulants every 2 days

  • Harvest cells after 7 days for comprehensive analysis

• Expression analysis methods:

  • Flow cytometry:

    • Use direct staining with markers (CD14, CD16, CD83, CD80)

    • Include FITC-conjugated LRRK2 antibody for direct detection

    • Gate populations to assess LRRK2 levels in specific subsets

  • Western blot analysis:

    • Lyse cells in appropriate buffer with protease inhibitors

    • Assess both mRNA (by qRT-PCR) and protein levels

    • Quantify both full-length and truncated LRRK2 forms

• Monitoring monocyte subtype transitions:

  • Track the shift from CD14⁺CD16⁻ to CD14⁺CD16⁺ cells after IFN-γ stimulation

  • Correlate this shift with changes in LRRK2 expression

  • Assess whether LRRK2 inhibitors (e.g., IN-1) attenuate this phenotypic transition

This protocol allows researchers to observe that IFN-γ robustly increases both LRRK2 mRNA and protein levels in monocytes, coinciding with their maturation from CD14⁺CD16⁻ to CD14⁺CD16⁺ phenotype—a process that can be modulated by LRRK2 inhibitors .

What methodological considerations are important when studying LRRK2's interaction with microtubules?

Studying LRRK2-microtubule interactions requires specialized approaches given their structural complexity:

• Structural analysis techniques:

  • Cryo-electron microscopy (cryo-EM) is the preferred method for visualizing LRRK2 bound to microtubules

  • Focus on the catalytic half containing kinase and GTPase domains, which adopts a closed conformation when bound to microtubules

  • Compare with LRRK1 (structurally similar but doesn't bind microtubules) to identify binding determinants

• Key binding domain identification:

  • The GTPase domain of LRRK2 mediates microtubule binding

  • Design site-directed mutagenesis experiments targeting specific amino acids in this domain

  • Validate mutants through in vitro binding assays and cellular localization studies

• Mutation impact assessment:

  • Test whether Parkinson's disease-linked mutations enhance microtubule binding

  • Determine if mutations affecting microtubule binding also alter kinase activity

  • Investigate whether altered microtubule binding correlates with pathogenicity

• Cellular localization studies:

  • Use fluorescently-labeled LRRK2 (or FITC-conjugated LRRK2 antibodies) to visualize co-localization with microtubules in cells

  • Apply confocal microscopy with super-resolution capabilities for detailed analysis

  • Assess whether kinase inhibitors affect LRRK2-microtubule association

• Kinase activity relationship:

  • Determine whether microtubule binding affects LRRK2 kinase activity

  • Test if kinase inhibitors influence microtubule binding

  • Investigate whether microtubule-bound LRRK2 has altered substrate specificity

Understanding these interactions has important implications for the design of therapeutic LRRK2 kinase inhibitors, as compounds that disrupt the LRRK2-microtubule interaction might have different effects than those that solely inhibit kinase activity .

How can researchers optimize immunofluorescence protocols for detecting LRRK2 in neuronal tissues?

Optimizing immunofluorescence for LRRK2 detection in neuronal tissues requires addressing several technical challenges:

• Tissue preservation and fixation:

  • For brain tissue: Use 4% paraformaldehyde fixation for 24-48 hours

  • For cultured neurons: Brief 10-minute fixation with 4% paraformaldehyde

  • Avoid over-fixation which can mask epitopes, especially when studying LRRK2 interactions with α-synuclein

• Antigen retrieval methods:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • For paraffin-embedded tissues: Additional proteinase K treatment may be necessary

  • Test multiple retrieval methods as LRRK2 epitopes can be fixation-sensitive

• Antibody selection and validation:

  • Use well-characterized antibodies (e.g., MJFF2 (c41-2))

  • Verify specificity with appropriate controls (LRRK2 knockout tissue/cells)

  • For co-localization studies, ensure antibodies are raised in different species to avoid cross-reactivity

• Signal amplification strategies:

  • Consider tyramide signal amplification for low-abundance detection

  • Use directly conjugated antibodies (FITC-LRRK2) to reduce background and cross-reactivity

  • For multi-labeling, carefully select fluorophores with minimal spectral overlap

• Specialized microscopy requirements:

  • Super-resolution techniques are recommended for detailed localization studies

  • For co-localization with α-synuclein in Lewy bodies, confocal z-stack imaging is essential

  • Use appropriate negative controls to establish thresholds for true co-localization

• Quantification approaches:

  • Employ digital image analysis with consistent parameters

  • Use Pearson's or Manders' coefficients for co-localization quantification

  • Develop region-specific analysis strategies, particularly for areas affected in Parkinson's disease

These optimized protocols enable researchers to accurately visualize LRRK2 distribution in neuronal tissues and identify its co-localization with other proteins of interest, particularly in pathological structures like Lewy bodies in Parkinson's disease patients .

What are common challenges when using FITC-conjugated antibodies for LRRK2 detection and how can they be addressed?

Researchers frequently encounter several technical challenges when working with FITC-conjugated LRRK2 antibodies:

• Photobleaching issues:

  • Problem: FITC fluorophore is particularly susceptible to photobleaching

  • Solution: Minimize exposure to light during all steps; use antifade mounting media; consider image acquisition with reduced illumination intensity and longer exposure times; process samples in darkened conditions

• Autofluorescence interference:

  • Problem: Neuronal tissues and blood cells often exhibit significant autofluorescence in the FITC spectrum

  • Solution: Include unstained controls; use spectral unmixing during image acquisition; treat samples with Sudan Black B or CuSO₄ to reduce autofluorescence; consider alternative conjugates with emission in different spectral ranges

• Signal-to-noise optimization:

  • Problem: Low signal-to-noise ratio when detecting endogenous LRRK2

  • Solution: Use signal amplification techniques; optimize antibody concentration through titration experiments; extend incubation times at 4°C; implement rigorous blocking protocols with species-matched serum

• Fixation artifacts:

  • Problem: Different fixation methods can affect FITC fluorescence and LRRK2 epitope accessibility

  • Solution: Compare multiple fixation protocols (paraformaldehyde, methanol, acetone); optimize fixation duration; consider mild fixation followed by post-fixation after antibody incubation

• Distinguishing specific from non-specific binding:

  • Problem: Determining true LRRK2 signal from background

  • Solution: Always include negative controls (isotype control, LRRK2 knockout/knockdown); perform blocking with both serum and BSA; include competitive binding controls with unlabeled antibody

• Antibody internalization during live-cell imaging:

  • Problem: FITC-conjugated antibodies may be internalized by live cells, complicating surface vs. internal protein distinction

  • Solution: Perform staining at 4°C to inhibit internalization; use membrane-impermeable crosslinkers to fix antibodies to surface proteins; compare with fixed cell controls

• pH sensitivity of FITC:

  • Problem: FITC fluorescence is pH-sensitive, potentially affecting signal in acidic cellular compartments

  • Solution: Maintain consistent pH during all experimental steps; consider pH-resistant fluorophore conjugates for studies involving lysosomes or other acidic compartments

Addressing these challenges through systematic optimization will significantly improve the reliability and interpretability of data obtained using FITC-conjugated LRRK2 antibodies.

How should researchers interpret conflicting data between different LRRK2 detection methods?

When faced with conflicting results between different LRRK2 detection methods, researchers should follow this systematic approach:

• Evaluate antibody specificity across methods:

  • Different antibodies may recognize distinct epitopes that are differentially accessible in various detection methods

  • Confirm results using multiple validated antibodies recognizing different LRRK2 domains

  • Compare results between C-terminal and N-terminal targeting antibodies to distinguish full-length vs. truncated forms

• Consider protein conformation effects:

  • Native vs. denatured conditions: Western blot (denatured) may detect epitopes hidden in native conformations used in flow cytometry or immunofluorescence

  • LRRK2's complex structure with multiple domains may adopt different conformations depending on activation state, affecting epitope accessibility

• Assess truncated forms and post-translational modifications:

  • The presence of both ~286 kDa (full-length) and ~170 kDa (truncated) LRRK2 forms can lead to discrepancies

  • Phosphorylation state (especially at Ser935) may affect antibody binding in certain assays

  • Treatment with LRRK2 inhibitors causes dephosphorylation that may alter detection patterns

• Analyze sample preparation differences:

  Detection Method	Sample Preparation	Potential Issues
  Western blot	Denatured protein	May detect epitopes hidden in native protein
  Flow cytometry	Cell fixation/permeabilization	Fixation can mask epitopes
  Immunofluorescence	Tissue fixation	Overfixation may reduce signal
  ELISA	Native or denatured protein	Buffer conditions may affect epitope availability

• Validate with functional assays:

  • Use LRRK2 kinase inhibitors (e.g., MLi-2) to confirm specificity of kinase activity measurements

  • Employ genetic approaches (siRNA, CRISPR) to validate antibody specificity

  • When studying phosphorylation events, compare with known LRRK2 substrates (e.g., Rab10)

• Consider context-dependent expression:

  • LRRK2 expression varies significantly between cell types (high in neutrophils, selective monocyte subpopulations)

  • Expression can be dynamically regulated (e.g., induction by IFN-γ in monocytes)

  • Activation state of cells may affect localization and detection

When reporting conflicting data, researchers should clearly describe methodological differences and consider publishing comprehensive validation data to advance the field's understanding of LRRK2 biology.

What factors affect the sensitivity and specificity of LRRK2 phosphorylation detection in clinical samples?

Detecting LRRK2 phosphorylation in clinical samples presents unique challenges that researchers must address:

• Pre-analytical variables:

  • Time between sample collection and processing: Phosphorylation states deteriorate rapidly

  • Temperature during collection and processing: Maintain samples at 4°C to minimize phosphatase activity

  • Anticoagulant choice for blood samples: EDTA preferred over heparin or citrate

  • Patient fasting status and medication use: Document and standardize when possible

• Phosphatase inhibition strategies:

  • Immediate addition of phosphatase inhibitor cocktails is critical

  • Include specific inhibitors of serine/threonine phosphatases

  • For neutrophils, incorporate DIFP to suppress intrinsic protease activity that could degrade phosphoproteins

  • Snap-freezing samples in liquid nitrogen when immediate processing isn't possible

• Antibody selection considerations:

  • Use phospho-specific antibodies validated with both phosphatase treatment and LRRK2 inhibitor controls

  • For Ser935 phosphorylation (indirect measure), confirm correlation with direct substrate phosphorylation

  • For direct substrate measurement, prioritize Rab10 Thr73 phosphorylation using MJFF-pRab10 antibody

• Cell type heterogeneity:

  • Neutrophils provide more homogeneous samples than PBMCs for LRRK2 analysis

  • When using mixed populations, employ cell-specific markers for accurate interpretation

  • Consider flow cytometry-based methods for single-cell resolution of phosphorylation status

• Signal normalization approaches:

  • Calculate phospho-to-total protein ratios rather than absolute phosphorylation levels

  • Include internal reference samples across batches for inter-assay normalization

  • Consider multiplex assays to simultaneously measure multiple phosphorylation sites

• Pathological considerations:

  • Disease state may affect baseline phosphorylation and response to inhibitors

  • G2019S LRRK2 mutation carriers may show different phosphorylation profiles

  • Inflammatory conditions can alter LRRK2 expression and phosphorylation patterns

Optimizing these factors can significantly improve the reliability of LRRK2 phosphorylation measurements in clinical samples, making them more suitable for diagnostic applications and pharmacodynamic studies of LRRK2 inhibitors .

How might FITC-conjugated LRRK2 antibodies contribute to developing biomarkers for Parkinson's disease?

FITC-conjugated LRRK2 antibodies offer several promising avenues for Parkinson's disease biomarker development:

• Flow cytometry-based peripheral blood assays:

  • Enable rapid quantification of LRRK2 expression levels in specific immune cell populations

  • Allow simultaneous assessment of multiple parameters (LRRK2 expression, phosphorylation state, and cell activation markers)

  • Potential for high-throughput screening in clinical settings with minimal sample processing

• Patient stratification applications:

  • Identify subgroups with altered LRRK2 pathway activity regardless of mutation status

  • Distinguish patients who might benefit from LRRK2 inhibitor therapies

  • Monitor disease progression through changes in LRRK2 expression or phosphorylation patterns

• Pharmacodynamic monitoring in clinical trials:

  • Direct measurement of target engagement for LRRK2 inhibitors

  • Quantification of downstream pathway effects through substrate phosphorylation

  • Real-time assessment of treatment response using peripheral blood neutrophils

• Integration with digital pathology platforms:

  • Automated image analysis of LRRK2 immunostaining in tissue biopsies

  • Machine learning algorithms to identify subtle changes in LRRK2 localization or expression

  • Correlation of LRRK2 patterns with clinical outcomes and disease progression

• Multi-parameter biomarker panels:

  • Combine LRRK2 measurements with other PD markers (α-synuclein, inflammatory mediators)

  • Develop algorithms incorporating clinical data with LRRK2 pathway activity

  • Longitudinal monitoring of multiple parameters to predict disease trajectory

The development of these biomarker approaches could significantly advance Parkinson's disease research by facilitating earlier diagnosis, more precise patient stratification, and objective measures of treatment efficacy, particularly for emerging LRRK2-targeted therapeutics .

What are the most promising approaches for studying LRRK2's role in immune cell function using fluorescently labeled antibodies?

Advanced immunological research on LRRK2 using fluorescently labeled antibodies presents several innovative approaches:

• Single-cell analysis of LRRK2 expression dynamics:

  • Apply flow cytometry with FITC-conjugated LRRK2 antibodies to track expression across immune cell subsets

  • Combine with cell lineage and activation markers to correlate LRRK2 levels with functional states

  • Monitor real-time changes in LRRK2 expression during immune cell differentiation and activation

• Spatial immune profiling in tissue microenvironments:

  • Implement multiplexed immunofluorescence to visualize LRRK2-expressing cells within tissue contexts

  • Map LRRK2 expression relative to inflammatory lesions in disease models

  • Correlate spatial distribution with disease progression markers and immune infiltration patterns

• Immune cell trafficking and migration studies:

  • Track LRRK2-expressing immune cells during recruitment to sites of inflammation

  • Assess whether LRRK2 inhibition affects immune cell mobilization and tissue infiltration

  • Investigate LRRK2's role in immune cell polarization and directional migration

• Mechanistic studies of monocyte maturation:

  • Monitor the IFN-γ-induced shift from CD14⁺CD16⁻ to CD14⁺CD16⁺ monocytes while tracking LRRK2 expression

  • Determine whether LRRK2 inhibitors (e.g., IN-1) directly modulate this phenotypic transition

  • Investigate downstream signaling pathways linking LRRK2 to monocyte differentiation

• Innate immune signaling pathway analysis:

  • Combine LRRK2 detection with phospho-flow cytometry to correlate LRRK2 with activation of specific signaling pathways

  • Assess LRRK2's role in pattern recognition receptor signaling

  • Determine how LRRK2 influences cytokine production profiles in specific immune cell subsets

These approaches leverage the specificity and direct visualization capabilities of fluorescently labeled LRRK2 antibodies to dissect the protein's role in immune function, potentially revealing new therapeutic targets at the intersection of neurodegeneration and inflammation .

How can advanced microscopy techniques enhance the utility of FITC-conjugated LRRK2 antibodies in neurodegenerative disease research?

Advanced microscopy techniques significantly expand the research applications of FITC-conjugated LRRK2 antibodies:

• Super-resolution microscopy approaches:

  • Structured Illumination Microscopy (SIM) improves resolution to ~120 nm for detailed LRRK2 localization

  • Stochastic Optical Reconstruction Microscopy (STORM) achieves ~20 nm resolution, revealing nanoscale LRRK2 organization

  • Stimulated Emission Depletion (STED) microscopy enables visualization of LRRK2 interaction with subcellular structures at ~50 nm resolution

• Live-cell imaging applications:

  • Spinning disk confocal microscopy allows real-time tracking of LRRK2 dynamics with minimal phototoxicity

  • Total Internal Reflection Fluorescence (TIRF) microscopy provides high-contrast imaging of LRRK2 near the plasma membrane

  • Photoactivation approaches to track LRRK2 movement between cellular compartments

• Correlative light and electron microscopy (CLEM):

  • Combine FITC-LRRK2 fluorescence with ultrastructural context

  • Precisely localize LRRK2 to subcellular structures like vesicles, microtubules, and mitochondria

  • Particularly valuable for studying LRRK2's association with pathological structures

• Multiplex imaging strategies:

  • Cyclic immunofluorescence for sequential imaging of numerous markers alongside LRRK2

  • Spectral unmixing to distinguish FITC-LRRK2 signal from tissue autofluorescence

  • Mass cytometry imaging (IMC) or co-detection by indexing (CODEX) for highly multiplexed tissue analysis

• Functional imaging applications:

  • Combine FITC-LRRK2 detection with calcium imaging to correlate localization with neuronal activity

  • FRET-based approaches to study LRRK2 protein-protein interactions in living cells

  • Optogenetic manipulation coupled with LRRK2 imaging to assess dynamic responses

• 3D brain mapping applications:

  • Light sheet microscopy for whole-tissue imaging of LRRK2 distribution

  • Tissue clearing techniques compatible with FITC-conjugated antibodies

  • 3D reconstruction of LRRK2 distribution relative to pathological hallmarks

These advanced microscopy approaches enable unprecedented insights into LRRK2's dynamic behavior, subcellular localization, and interaction with disease-relevant structures such as α-synuclein-containing Lewy bodies or microtubule networks .