LRRTM4 Antibody, HRP conjugated

What is LRRTM4 and what is its role in neural systems?

LRRTM4 is a trans-synaptic adhesion protein that plays critical roles in neural development and synaptic function. It belongs to the leucine-rich repeat transmembrane (LRRTM) family and is expressed predominantly in neuronal tissues. The protein contains 10 leucine-rich repeat (LRR) domains which form a characteristic horseshoe fold structure accommodating leucine residues within a tightly packed core .

LRRTM4 functions in both synaptogenesis and synaptic maintenance. In the central nervous system, it regulates glutamatergic synapse assembly on dendrites of central neurons . Interestingly, in the retina, LRRTM4 is enriched at GABAergic synapses on axon terminals of rod bipolar cells (RBCs), where it influences inhibitory synapse organization and function . It exhibits strong synaptogenic activity that is primarily restricted to excitatory presynaptic differentiation .

Recent research has revealed that LRRTM4 is a 590 amino acid single-pass type I membrane protein that contributes to the development and maintenance of the vertebrate nervous system . It exists in at least two isoforms produced by alternative splicing, with differential expression patterns in various neural tissues .

What applications are LRRTM4 antibodies suitable for?

LRRTM4 antibodies, particularly HRP-conjugated variants, are versatile research tools applicable to multiple experimental techniques:

LRRTM4 antibodies have been successfully used in immunohistochemical analyses of human brain tissue and in visualizing the protein at rod bipolar cell dendritic tips in retinal tissues . For optimal results in Western blotting, mouse brain lysate has proven to be an effective positive control sample .

What is the cellular and subcellular localization of LRRTM4?

LRRTM4 demonstrates distinct localization patterns that vary by neural tissue type:

In the retina, LRRTM4 shows remarkable specificity:

• Predominantly localized at rod bipolar cell (RBC) terminals

• Enriched at GABAergic synapses on RBC axon terminals

• Present at rod BC dendritic tips, where it colocalizes with the transduction channel protein TRPM1

• Notably absent from ON-cone bipolar cell terminals and dendritic tips

In other neural tissues:

• LRRTM4 is primarily expressed in neuronal tissues across the central nervous system

• Functions at the cell membrane as a single-pass type I membrane protein

• In the hippocampus, LRRTM4 regulates glutamatergic (AMPA receptor-driven) synapses on dentate gyrus granule cells

• Similarly affects glutamatergic synapses on L2/3 cortical neurons

This differential distribution underscores LRRTM4's tissue-specific roles in synapse organization and function across diverse neural circuits.

What are the optimal immunostaining protocols for LRRTM4 antibody, HRP conjugated?

For optimal immunostaining results with HRP-conjugated LRRTM4 antibodies, consider the following protocol recommendations:

Paraffin-Embedded Tissue Protocol:

• Deparaffinize sections through xylene and graded alcohols

• Perform heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes

• Block endogenous peroxidase activity with 3% H₂O₂ in methanol for 15 minutes

• Apply protein blocking solution (1% BSA, 0.3% Triton X-100 in PBS) for 1 hour at room temperature

• Incubate with LRRTM4 HRP-conjugated antibody at 1:100 dilution overnight at 4°C

• Since the antibody is directly HRP-conjugated, no secondary antibody is required

• Develop with DAB substrate and counterstain with hematoxylin

• Dehydrate, clear, and mount with permanent mounting medium

Frozen Section Protocol:

• Fix sections in cold 4% paraformaldehyde for 10 minutes

• Wash thoroughly with PBS (3 × 5 minutes)

• Block with 5% normal serum, 0.3% Triton X-100 in PBS for 1 hour at room temperature

• Incubate with LRRTM4 HRP-conjugated antibody at 1:100-1:200 dilution for 2 hours at room temperature or overnight at 4°C

• Wash thoroughly (3 × 10 minutes PBS)

• Develop with DAB substrate

• Counterstain as needed and mount with appropriate medium

These protocols have been optimized based on experimental evidence with LRRTM4 antibodies in neural tissues and provide a methodological foundation that can be further optimized for specific experimental conditions .

How does LRRTM4 regulate GABAergic synapses in retinal rod bipolar cells?

LRRTM4's regulation of GABAergic synapses in rod bipolar cells (RBCs) represents a fascinating departure from its more commonly studied role in glutamatergic synapse organization. The regulatory mechanism involves several key aspects:

• Receptor Clustering: LRRTM4 is essential for proper clustering of both GABA₁₁ and GABA_C receptors at RBC terminals. Knockout of LRRTM4 significantly reduces the expression of these receptors at GABAergic synapses .

• Functional Impact: Electrophysiological studies using whole-cell patch clamp recordings demonstrate that LRRTM4 knockout substantially reduces:

  • Total GABA-evoked inhibitory currents

  • GABA_A-mediated current component

  • GABA_C-mediated current component

• Developmental Aspects: The effects of LRRTM4 on GABAergic synapse organization are present from early development (P12) through adulthood, suggesting a role in both synapse formation and maintenance .

• Structural Organization: LRRTM4 influences the stereotyped arrangement of synaptic dyads at RBC terminals. In normal RBCs, synaptic ribbons are apposed to two distinct postsynaptic partners (forming a "dyad"). In LRRTM4-deficient retinas, abnormal "monad" and "triad" arrangements emerge .

This regulatory mechanism appears to involve interactions with the extracellular matrix, as LRRTM4's extracellular domain engages in heparan-sulfate dependent binding with proteins like pikachurin . These findings highlight LRRTM4's multifaceted role in organizing different types of synapses in a circuit-specific manner.

What controls and validation steps are essential when using LRRTM4 antibodies?

Rigorous validation of LRRTM4 antibodies is critical for experimental reliability. Implement these essential controls and validation procedures:

Validation Steps:

• Knockout/Knockdown Controls:

  • Compare antibody labeling between wildtype tissue and LRRTM4 knockout/knockdown samples

  • CRISPR/Cas9-mediated somatic knockout in bipolar cells has been successfully used to validate LRRTM4 antibodies

  • Confirmation should show abolished or significantly reduced antibody signal in knockout tissues

• Expression Pattern Verification:

  • LRRTM4 shows cell-specific expression patterns (e.g., present in rod bipolar cells but not ON-cone bipolar cells)

  • Verify expected cellular distribution in your tissue of interest

  • Cross-reference with established expression data from transcriptomic studies

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess immunizing peptide prior to immunostaining

  • Signal should be significantly reduced or eliminated if the antibody is specific

• Western Blot Validation:

  • Confirm detection of a single band at the expected molecular weight (67 kDa)

  • Mouse brain lysate serves as an effective positive control

  • Test multiple tissue types to confirm tissue-specific expression patterns

• Co-localization Studies:

  • Verify co-localization with established markers:

    • In retina: PKC (for RBC identification)

    • At synapses: GABA₁₁ or TRPM1 (depending on the neural circuit)

• Cross-Antibody Validation:

  • Compare labeling patterns using antibodies targeting different epitopes of LRRTM4

  • Consistent patterns across different antibodies increase confidence in specificity

These validation approaches have been demonstrated effective in published research on LRRTM4 and constitute best practices for ensuring experimental rigor when working with LRRTM4 antibodies .

How can researchers design experiments to investigate LRRTM4's isoform-specific functions?

Designing experiments to distinguish and characterize LRRTM4 isoform-specific functions requires a multifaceted approach:

Experimental Design Strategies:

• Isoform-Specific qPCR Analysis:

  • Design primers that specifically amplify each LRRTM4 isoform

  • Quantify relative expression levels across different neural tissues

  • Previous studies have shown that the short isoform of LRRTM4 is enriched in both retina and other brain regions relative to the long isoform

• Isoform-Selective Antibodies:

  • Utilize antibodies targeting regions unique to each isoform

  • The immunogen range of commercially available antibodies should be verified against isoform-specific sequences (e.g., bs-11878R-HRP targets region 135-250/590)

  • Perform Western blot analysis to confirm detection of different molecular weight bands corresponding to each isoform

• Expression Constructs for Functional Studies:

  Construct	Purpose	Analysis Method
  Full-length isoforms	Rescue experiments in knockout backgrounds	Immunocytochemistry, electrophysiology
  Domain-specific mutants	Identify critical functional regions	Binding assays, synaptogenesis assays
  Fluorescently tagged constructs	Subcellular localization	Live imaging, FRAP

• Isoform-Specific Knockdown/Knockout:

  • Design CRISPR/Cas9 or shRNA approaches targeting isoform-specific sequences

  • CRISPR/Cas9 somatic knockout in BCs by subretinal injection and electroporation has been successfully employed

  • Compare phenotypes between isoform-specific and complete LRRTM4 knockout

• Binding Partner Analysis:

  • Perform pull-down assays using recombinant isoform-specific proteins

  • Test binding to known partners like pikachurin, which engages in heparan-sulfate dependent binding with LRRTM4's extracellular domain

  • Use surface plasmon resonance to quantify binding affinities of different isoforms

• Functional Rescue Experiments:

  • In LRRTM4 knockout systems, express individual isoforms and assess:

    • Recovery of GABA receptor clustering in RBCs

    • Restoration of normal synaptic dyad arrangements

    • Rescue of GABA-evoked inhibitory currents

These approaches provide a comprehensive framework for dissecting isoform-specific functions of LRRTM4 across different neural systems, building on established methodologies from published research .

What are common troubleshooting steps for weak or non-specific LRRTM4 antibody signals?

When encountering issues with LRRTM4 antibody signal, consider these evidence-based troubleshooting approaches:

For Weak Signal:

• Epitope Retrieval Optimization:

  • Extend heat-induced epitope retrieval time (20-30 minutes)

  • Test alternative retrieval buffers (citrate pH 6.0 vs. EDTA pH 9.0)

  • For some neural tissues, proteolytic retrieval with proteinase K may improve signal

• Antibody Concentration Adjustment:

  • Increase antibody concentration (try 1:50 dilution for IHC applications)

  • Extend incubation time to overnight at 4°C

  • For HRP-conjugated antibodies, ensure H₂O₂ quenching is complete to maximize signal

• Signal Amplification:

  • For unconjugated primary antibodies, employ tyramide signal amplification

  • Use higher sensitivity detection systems (polymer-based vs. ABC method)

  • Consider specialized enhancement systems for HRP-conjugated antibodies

For Non-specific Signal:

• Blocking Optimization:

  • Increase blocking duration (2-3 hours)

  • Test different blocking agents (5% normal serum, 1% BSA, commercial blockers)

  • Add 0.3% Triton X-100 to reduce background in neural tissues

• Washing Protocols:

  • Extend washing steps (5 × 10 minutes)

  • Use higher salt concentration in wash buffer (PBS with 0.5M NaCl)

  • Add 0.1% Tween-20 to wash buffers

• Antibody Validation:

  • Test antibody on known positive and negative control tissues

  • Mouse brain tissue serves as an excellent positive control

  • Tissues from LRRTM4 knockout animals provide ideal negative controls

• Fixation Considerations:

  • Optimize fixation time (over-fixation can mask epitopes)

  • For fresh frozen sections, test brief post-fixation (10 minutes 4% PFA)

  • For cultured cells, compare methanol vs. paraformaldehyde fixation

Each of these approaches addresses specific aspects of immunodetection that might affect LRRTM4 antibody performance, based on demonstrated approaches in published research with LRRTM4 antibodies .

How can researchers design multiplexed experiments studying LRRTM4 with other synaptic markers?

Multiplexed detection of LRRTM4 alongside other synaptic proteins enables comprehensive analysis of complex synaptic arrangements. Here are methodological considerations for successful multiplexed experiments:

Antibody Selection and Validation:

• Host Species Compatibility:

  • Select primary antibodies from different host species to avoid cross-reactivity

  • For example, use rabbit anti-LRRTM4 with mouse anti-PKC for RBC identification

  • When using HRP-conjugated LRRTM4 antibody, pair with fluorescently-labeled antibodies for other targets

• Sequential Detection Protocols:

  • For multi-round labeling with HRP-conjugated antibodies:

    • Develop first HRP-antibody with DAB/nickel (black)

    • Strip or quench HRP activity completely

    • Apply second HRP-conjugated antibody

    • Develop with DAB only (brown)

  • Alternatively, use tyramide-based sequential immunofluorescence

Recommended Marker Combinations:

Technical Approaches:

• Multicolor Confocal Microscopy:

  • Use spectrally separated fluorophores (e.g., Alexa 488, Cy3, Cy5)

  • Employ sequential scanning to minimize bleed-through

  • For retinal tissues, optical sectioning at 0.3-0.5μm intervals captures synaptic detail

• Array Tomography:

  • For high-resolution multi-protein localization

  • Serial ultrathin sections (70-200nm) on silicon chips

  • Multiple rounds of antibody staining/elution

  • Enables detection of 10+ proteins at the same synapses

• Expansion Microscopy:

  • Physically expand tissue to improve resolution

  • Particularly useful for crowded synaptic regions

  • Compatible with multiple rounds of staining

• High-Content Imaging Analysis:

  • Automated analysis of protein colocalization

  • Quantification of synaptic protein clustering

  • Statistical evaluation of spatial relationships

These approaches have proven effective in LRRTM4 research, particularly in studies examining its relationship with synaptic partners in the retina and other neural tissues .

What emerging techniques show promise for studying LRRTM4 function in neural circuits?

Several cutting-edge methodologies offer new possibilities for investigating LRRTM4's role in neural circuit organization and function:

Advanced Genetic Approaches:

• Cell-Type Specific Conditional Knockouts:

  • Cre-loxP systems targeting specific neuronal populations

  • Allows assessment of LRRTM4 function in defined circuits while avoiding developmental compensation

  • Particularly valuable given LRRTM4's differential expression between rod bipolar cells and ON-cone bipolar cells

• CRISPR/Cas9-Based Strategies:

  • Somatic CRISPR knockout via subretinal injection and electroporation has already proven effective

  • Next-generation base editors for introducing specific mutations

  • CRISPRa/CRISPRi for reversible modulation of LRRTM4 expression

Protein Interaction Analysis:

• Proximity Labeling Techniques:

  • BioID or TurboID fusion with LRRTM4 to identify proximal proteins

  • APEX2-based labeling for electron microscopy visualization

  • Split-BioID for detecting specific protein-protein interactions in situ

• High-Resolution Structural Studies:

  • Cryo-EM analysis of LRRTM4 complexes with binding partners

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

  • Particularly informative for understanding LRRTM4's heparan-sulfate dependent binding with pikachurin

Functional Imaging Technologies:

• Super-Resolution Microscopy:

  • STORM/PALM imaging of LRRTM4 nanoscale organization

  • Live-cell super-resolution to track dynamic changes

  • Multicolor STORM to visualize LRRTM4 with interacting partners

• Functional Connectomics:

  • Serial-section electron microscopy with immunogold labeling

  • Correlative light and electron microscopy (CLEM)

  • Particularly valuable for examining LRRTM4's role in organizing synaptic dyads at RBC terminals

• Optogenetic/Chemogenetic Approaches:

  • Combining cell-specific manipulation with LRRTM4 knockout

  • Assessing circuit-level consequences of LRRTM4 deficiency

  • Particularly relevant for understanding LRRTM4's impact on presynaptic inhibition in the retina

These emerging approaches promise to advance our understanding of LRRTM4's multifaceted roles in neural circuit development and function, building upon foundational discoveries made with traditional techniques while providing unprecedented resolution and specificity.

How can researchers design experiments to resolve contradictory findings about LRRTM4 function?

When confronted with contradictory findings about LRRTM4 function across different neural systems, researchers can employ systematic experimental approaches to reconcile discrepancies:

Comparative Analysis Framework:

• Cross-Tissue Systematic Comparison:

  • Design parallel experiments examining LRRTM4 in multiple neural systems:

    • Retina (GABAergic function at RBC terminals)

    • Hippocampus (glutamatergic function in dentate gyrus)

    • Cortex (L2/3 neurons)

  • Use identical methodologies and reagents across systems

  • Quantify differences in LRRTM4 expression levels, binding partners, and functional impacts

• Isoform-Specific Resolution:

  • Determine if contradictions stem from differential isoform expression

  • The short isoform of LRRTM4 is enriched in retina and other brain regions

  • Design isoform-specific knockdown/rescue experiments

  • Create comprehensive isoform expression maps across neural tissues

Methodological Harmonization:

• Standardized Protein Interaction Studies:

  • Use consistent methodologies to examine LRRTM4 binding partners:

    Technique	Application	Advantage
    Co-immunoprecipitation	Verify protein interactions	Preserves native complexes
    Surface plasmon resonance	Measure binding kinetics	Quantitative comparison
    Proximity ligation assay	In situ interaction detection	Maintains tissue context

  • Apply identical assay conditions across neural systems

  • Compare LRRTM4's interactions with both GABAergic and glutamatergic components

• Developmental Timeline Analysis:

  • Track LRRTM4 expression and function across developmental stages

  • Determine if contradictory findings reflect temporal differences

  • LRRTM4's effects on GABAergic synapses are present from P12 through adulthood

  • Establish parallel developmental timelines across neural systems

Advanced Integrative Approaches:

• Domain-Function Mapping:

  • Create a series of chimeric constructs swapping domains between LRRTM family members

  • Identify which domains confer tissue-specific functions

  • Map critical regions for GABAergic versus glutamatergic functions

  • Perform structure-function analysis of LRRTM4's 10 LRR domains

• Proteomics and Interactome Analysis:

  • Perform unbiased pull-downs coupled with mass spectrometry

  • Compare LRRTM4 interactomes across neural systems

  • Identify tissue-specific binding partners

  • Connect interactome differences to functional variations

• Multi-modal Circuit Analysis:

  • Combine electrophysiology, imaging, and behavioral analysis

  • Assess how LRRTM4 manipulation affects circuit function

  • Compare consequences across different neural systems

  • Link molecular interactions to circuit-level outcomes

By implementing these systematic approaches, researchers can resolve apparent contradictions about LRRTM4 function, revealing whether differences represent genuine biological diversity in LRRTM4's roles or stem from methodological variations across studies .

What are the optimal quantification approaches for analyzing LRRTM4 expression and colocalization?

Rigorous quantitative analysis of LRRTM4 immunostaining requires appropriate methodologies tailored to the specific research questions:

Expression Quantification:

Colocalization Analysis:

• Object-Based Colocalization:

  • Ideal for punctate synaptic proteins like LRRTM4

  • Identify discrete LRRTM4 puncta and determine overlap with markers like GABA₁₁ or TRPM1

  • Quantify parameters including:

    • Percent of LRRTM4 puncta colocalized with partner protein

    • Distance between puncta centers

    • Size and intensity of colocalized versus non-colocalized puncta

• Pixel-Based Colocalization:

  • Calculate standard coefficients:

    • Pearson's correlation coefficient

    • Manders' overlap coefficient

    • Intensity correlation quotient

  • Perform on raw confocal data after chromatic aberration correction

  • Use randomization tests to establish significance

• Line Profile Analysis:

  • Draw line profiles across synaptic structures

  • Plot normalized intensities of LRRTM4 and partner proteins

  • Assess peak alignment and relative distributions

  • Particularly useful for examining LRRTM4 distribution relative to pre- and post-synaptic markers

Statistical Considerations:

• Use appropriate statistical tests based on data distribution

• For volume occupancy comparisons between LRRTM4 knockout and control, t-tests or Mann-Whitney tests are appropriate

• For multiple condition comparisons, employ ANOVA with appropriate post-hoc tests

• Report effect sizes alongside p-values

• Present data using scatter plots showing individual data points alongside means and error bars

These quantification approaches have proven effective in published LRRTM4 research and provide a rigorous framework for analyzing immunolabeling data in various experimental contexts .

What controls are necessary for interpreting electrophysiological data in LRRTM4 studies?

When conducting electrophysiological studies to investigate LRRTM4 function, particularly in contexts like GABA receptor function in retinal bipolar cells, the following controls and considerations are critical:

Recording Quality Controls:

• Cell Health Verification:

  • Monitor series resistance throughout recordings

  • Verify stable input resistance and holding current

  • Document cell morphology and position

  • Particularly important when recording from LRRTM4 KO cells which may have altered properties

• Consistent Stimulus Parameters:

  • For GABA-evoked responses:

    • Standardize GABA concentration (typically 100 μM for retinal studies)

    • Control puff duration and pressure

    • Maintain consistent puff pipette positioning relative to RBC terminals

  • Document all stimulus parameters for reproducibility

Pharmacological Controls:

• Receptor Subtype Discrimination:

  • Apply specific antagonists sequentially:

    • TPMPA for GABA_C receptor blockade

    • GABAzine for GABA_A receptor blockade

  • Verify complete blockade with combined antagonists

  • Compare receptor component ratios between experimental conditions

• Isolating Specific Currents:

  • Block non-GABA receptors with appropriate antagonists

  • Control for potential indirect effects through other circuits

  • Verify specificity of observed effects through pharmacological occlusion tests

Experimental Design Controls:

• Age-Matched Comparisons:

  • Use littermate controls for LRRTM4 KO studies

  • Match developmental stages precisely (P12 vs. adult)

  • Document age-dependent effects with appropriate developmental series

• Cell-Type Verification:

  • Confirm recorded cell identity (RBCs vs. ON-CBCs)

  • Include morphological documentation

  • Consider using fluorescently labeled transgenic mouse lines (e.g., Grm6-tdtomato)

Analysis Guidelines: