LRRTM4 Antibody, FITC conjugated

What is LRRTM4 and what are its primary functions in neural tissues?

LRRTM4 functions as a trans-synaptic adhesion protein that regulates glutamatergic synapse assembly on dendrites of central neurons. In the mouse retina, LRRTM4 is distinctly enriched at GABAergic synapses on axon terminals of rod bipolar cells (RBCs) . The protein exists in multiple isoforms, with the short isoform being predominantly expressed in the retina relative to the long isoform, similar to its expression pattern in other brain regions . Functionally, LRRTM4 plays a critical role in maintaining GABA receptor expression at RBC terminals, as evidenced by significant downregulation of both GABA Aα1 and GABA C receptors in LRRTM4 knockout mouse models .

What cellular localization pattern does LRRTM4 typically display when detected with antibodies?

When detected with specific antibodies, LRRTM4 displays distinct localization patterns that vary by cell type. In the retina, LRRTM4 immunoreactivity is predominantly localized at rod bipolar cell (RBC) terminals with relatively little signal at ON cone bipolar cell terminals . More specifically, LRRTM4 is found at GABAergic synapses on RBC axons. Research using 3D confocal image stack analysis reveals that LRRTM4 puncta at RBC terminals not colocalized with GABA Aα1 are likely localized at GABA C synapses . This distinctive distribution pattern suggests specialized functional roles at inhibitory synapses in the retinal circuitry.

How should FITC-conjugated antibodies be stored to maintain optimal fluorescence activity?

For FITC-conjugated antibodies including those targeting LRRTM4, proper storage is critical to preserve fluorescence activity. Lyophilized antibodies should be stored at +4°C prior to reconstitution . After reconstitution (typically adding 50 μl H₂O to achieve a 1mg/ml solution in PBS), the antibody should be aliquoted and stored at -20°C to -80°C until use . It is important to note that freezing should be avoided when the antibody is still in lyophilized form . Additionally, FITC conjugates are particularly sensitive to photobleaching, so all storage vessels should be wrapped in aluminum foil or kept in light-protected containers, and extended exposure to light during experimental procedures should be minimized.

What are the optimal conditions for FITC conjugation to LRRTM4 antibodies?

The conjugation of FITC to antibodies including those against LRRTM4 requires careful optimization of reaction conditions to achieve ideal labeling efficiency. Research indicates that maximal labeling is achieved under the following conditions:

• Reaction time: 30-60 minutes

• Temperature: Room temperature

• pH: 9.5

• Initial protein concentration: 25 mg/ml

Additionally, using relatively pure IgG obtained by DEAE Sephadex chromatography and high-quality FITC is essential for optimal conjugation . The molecular fluorescein/protein (F/P) ratio is a critical parameter to monitor, as both under-labeled and over-labeled antibodies may have reduced performance. Separation of optimally labeled antibodies from under- and over-labeled proteins can be effectively achieved using gradient DEAE Sephadex chromatography .

What controls should be included when performing immunofluorescence with FITC-conjugated LRRTM4 antibodies?

When conducting immunofluorescence studies with FITC-conjugated LRRTM4 antibodies, several controls are essential to ensure specificity and accuracy:

• Negative controls: Include samples from LRRTM4 knockout animals when available . This provides definitive evidence of antibody specificity.

• Blocking controls: Pre-block fixed/permeabilized cells with unlabeled LRRTM4 antibody prior to staining with the FITC-conjugated antibody to demonstrate staining specificity .

• Isotype controls: Use appropriate isotype control antibodies (e.g., rat IgG1 for rat-derived antibodies) at comparable concentrations to assess background staining levels .

• Positive controls: Include tissues known to express LRRTM4, such as mouse retina, particularly rod bipolar cell terminals .

• Internal controls: Co-label with markers unaffected by LRRTM4 expression, such as the ribbon marker C-terminal binding protein-2 (CtBP2), which shows comparable expression regardless of LRRTM4 status .

How can FITC-conjugated LRRTM4 antibodies be used to investigate GABAergic synapse formation in the retina?

FITC-conjugated LRRTM4 antibodies provide a powerful tool for investigating GABAergic synapse formation in the retina, particularly at rod bipolar cell (RBC) terminals. Based on research findings, a comprehensive protocol might include:

• Tissue preparation: Use paraformaldehyde-fixed retinal sections or whole-mounts depending on the research question.

• Co-labeling strategy: Combine FITC-conjugated LRRTM4 antibodies with markers for:

  • RBCs (using PKC antibodies)

  • GABA receptors (GABA Aα1 and GABA C receptor antibodies)

  • Synaptic ribbons (CtBP2 antibody)

• Quantitative analysis: Measure percent volume occupancy of GABA receptor subtypes within RBC terminals as a function of LRRTM4 expression . This can be accomplished through 3D confocal microscopy followed by image analysis to determine colocalization coefficients.

• Functional correlation: Combine immunofluorescence data with electrophysiological recordings of GABA-evoked responses to establish structure-function relationships .

This approach has successfully demonstrated that LRRTM4 knockout leads to reduced GABA Aα1 and GABA C receptor expression within RBC terminals, correlating with diminished GABA-evoked currents measured by electrophysiology .

What techniques can be used to distinguish between different LRRTM4 isoforms using antibodies?

Distinguishing between LRRTM4 isoforms (short and long) requires specialized approaches:

• Isoform-specific antibodies: Design or select antibodies that target unique epitopes present in only one isoform. The immunogen selection is critical - for example, an antibody raised against amino acids 446-517 of mouse LRRTM4 (UniProt Id: Q80XG9) may detect specific isoforms depending on the sequence conservation .

• Validation strategy:

  • Perform Western blotting to confirm band sizes corresponding to different isoforms

  • Use tissues with known isoform expression patterns as positive controls (e.g., retina predominantly expresses the short isoform)

  • Include samples from LRRTM4 knockout animals as negative controls

• Combined approaches: Complement antibody-based detection with molecular techniques such as real-time quantitative PCR to confirm isoform expression, as demonstrated in studies showing enrichment of the short LRRTM4 isoform in mouse retina .

• Subcellular localization analysis: Map the distribution patterns of different isoforms using high-resolution microscopy (confocal or super-resolution), as isoforms may localize to different subcellular compartments or synapse types.

How can LRRTM4 antibodies be used in investigating potential roles of LRRTM4 in cancer research?

Recent research has identified connections between LRRTM4 and cancer biology, particularly through a long noncoding RNA termed lnc-LRRTM4. FITC-conjugated LRRTM4 antibodies can be valuable tools in cancer research through these approaches:

• Expression profiling: Determine LRRTM4 protein expression levels in colorectal cancer (CRC) tissues compared to normal tissues using immunofluorescence microscopy or flow cytometry .

• Mechanism investigation: Study the relationship between lnc-LRRTM4 and LRRTM4 protein expression through combined immunofluorescence and RNA FISH (Fluorescence In Situ Hybridization) techniques .

• Functional analysis: Use LRRTM4 antibodies to monitor changes in LRRTM4 protein levels following experimental manipulation of lnc-LRRTM4 expression in cancer cell lines .

• Cellular process assessment: Investigate the role of LRRTM4 in epithelial-mesenchymal transition (EMT) by analyzing co-expression with EMT markers in CRC tissues .

Research has shown that lnc-LRRTM4 promotes the proliferation and metastasis of CRC cells by directly binding to the promoter of LRRTM4 to induce its transcription . FITC-conjugated LRRTM4 antibodies would allow for direct visualization of this relationship in tissue samples.

What strategies can address weak LRRTM4 immunofluorescence signal when using FITC-conjugated antibodies?

When encountering weak signals with FITC-conjugated LRRTM4 antibodies, consider the following optimization strategies:

• Antibody concentration optimization: Titrate the antibody to determine optimal concentration. For immunofluorescent staining in flow cytometry applications, ≤0.5 μg antibody per million cells is often recommended as a starting point .

• Signal amplification methods:

  • Use a biotin-streptavidin system with a biotinylated primary antibody and FITC-conjugated streptavidin

  • Employ tyramide signal amplification for significantly enhanced sensitivity

• Fixation optimization: Test different fixation protocols, as overfixation can mask epitopes while underfixation may compromise tissue morphology.

• Antigen retrieval: Implement heat-induced or enzymatic antigen retrieval methods to expose epitopes that might be masked during fixation.

• Permeabilization adjustment: Optimize detergent type and concentration for adequate antibody access to intracellular epitopes.

• Minimize photobleaching: FITC is particularly susceptible to photobleaching; use anti-fade mounting media and minimize exposure to light during all procedures.

• F/P ratio consideration: Ensure the antibody has an optimal fluorescein/protein ratio, typically between 3:1 and 6:1, as both over-labeling and under-labeling can compromise performance .

How can FITC-conjugated LRRTM4 antibodies be integrated into multicolor immunofluorescence panels?

Integrating FITC-conjugated LRRTM4 antibodies into multicolor panels requires careful consideration of spectral overlap and staining strategy:

• Spectral compatibility: FITC (excitation ~495 nm, emission ~520 nm) works well with fluorophores such as:

  • Cy3 or TRITC (minimal spectral overlap with FITC)

  • APC (far-red spectrum, minimal overlap)

  • DAPI (for nuclear counterstaining)

• Panel design considerations:

  • Reserve FITC for antigens expressed at lower levels when possible, as FITC has moderate brightness compared to newer fluorophores

  • Consider compensation requirements when using flow cytometry

• Sequential staining approach:

  • Apply primary antibodies sequentially rather than in a cocktail to minimize cross-reactivity

  • Begin with the FITC-conjugated LRRTM4 antibody, as FITC is more susceptible to photobleaching

• Validation strategy:

  • Include single-color controls for each fluorophore

  • Use fluorescence-minus-one (FMO) controls to establish accurate gating when using flow cytometry

• Imaging considerations:

  • Use sequential scanning on confocal microscopes to prevent bleed-through

  • Optimize exposure settings for each channel individually

These strategies have been successfully employed in studies involving GABA receptors and synaptic proteins in the retina, where multiple markers were simultaneously visualized .

How might FITC-conjugated LRRTM4 antibodies contribute to understanding neuropsychiatric disorders?

LRRTM4's role in synaptic organization suggests potential implications for neuropsychiatric conditions. FITC-conjugated LRRTM4 antibodies could contribute to this field through:

• Comparative expression studies: Analyze LRRTM4 expression patterns in postmortem brain tissues from patients with neuropsychiatric disorders versus controls.

• Animal model investigations: Examine LRRTM4 distribution in genetic models of autism spectrum disorders, schizophrenia, or intellectual disability where synaptic dysfunction is implicated.

• Drug screening applications: Use LRRTM4 immunofluorescence as a readout for high-content screening of compounds that modulate synapse formation or maintenance.

• Circuit-specific analysis: Combine FITC-conjugated LRRTM4 antibodies with markers for specific neural circuits implicated in neuropsychiatric conditions to identify circuit-specific alterations in LRRTM4 expression or localization.

• Developmental studies: Track LRRTM4 expression during critical periods of brain development that may be vulnerable to disruption in neuropsychiatric disorders.

The selective expression of LRRTM4 at specific synapses, such as its enrichment at GABAergic synapses on rod bipolar cell terminals , suggests that understanding its distribution in other brain regions could provide insights into circuit-specific vulnerabilities in neuropsychiatric conditions.

What experimental approaches can combine LRRTM4 immunolabeling with modern tissue clearing techniques?

Combining FITC-conjugated LRRTM4 antibodies with advanced tissue clearing methods offers powerful opportunities for whole-tissue imaging:

• Compatible clearing protocols:

  • CLARITY or PACT (passive CLARITY) preserve fluorescent proteins and are compatible with immunostaining

  • iDISCO+ allows for whole-organ immunolabeling followed by clearing

  • CUBIC provides good transparency and protein retention

• Methodological considerations:

  • Perform antibody validation in thin sections before proceeding to whole-tissue immunolabeling

  • Optimize antibody concentration and incubation time for thick tissue penetration

  • Consider using fragment antibodies (Fab) for better penetration in dense tissues

• Sequential labeling strategy:

  • Apply LRRTM4 antibodies early in the staining sequence

  • Use prolonged incubation times (days to weeks) depending on tissue thickness

  • Employ constant gentle agitation to enhance antibody penetration

• Analysis approaches:

  • Light-sheet microscopy for rapid imaging of large cleared volumes

  • Confocal microscopy with long working-distance objectives for detailed analysis of regions of interest

  • 3D reconstruction software for visualizing LRRTM4 distribution throughout neural circuits

These approaches could be particularly valuable for mapping LRRTM4 distribution throughout the entire retinal network or across brain regions, extending beyond the focused analyses of rod bipolar cell terminals described in current literature .