LRIT1 Antibody, FITC conjugated

What is LRIT1 and why is it significant in neuroscience research?

LRIT1 is a transmembrane protein primarily expressed in photoreceptor cells of the retina. It plays a crucial role in synaptic communication between photoreceptors and bipolar cells. Research has demonstrated that LRIT1 modulates adaptive changes in synaptic communication of cone photoreceptors, making it significant for understanding daylight vision mechanisms . LRIT1 has been shown to engage in trans-synaptic interactions with mGluR6, the principal receptor in postsynaptic ON-bipolar cells, contributing to the regulation of visual signal processing .

What are the structural characteristics of LRIT1 protein?

LRIT1 contains leucine-rich repeat domains, an immunoglobulin-like domain, and a transmembrane domain. The protein forms homodimers and/or heterodimers with related proteins such as LRIT2. Importantly, immunoprecipitation studies have shown that LRIT1 interacts with Frmpd2 via its intracellular domain, specifically through the third PDZ domain (PDZ3) of Frmpd2 . The extracellular domain contains the epitopes typically targeted by commercially available antibodies.

What does a FITC conjugation provide to LRIT1 antibodies?

FITC (Fluorescein Isothiocyanate) conjugation enables direct visualization of LRIT1 protein in samples without requiring a secondary antibody step. The conjugation provides:

• Direct detection capability with excitation/emission maxima at 495 nm/524 nm

• A bright green fluorescence signal compatible with standard FITC filter sets

• Compatibility with multiple immunofluorescence protocols including tissue sections and cell cultures

• Reduced protocol time by eliminating secondary antibody incubation and washing steps

What are the recommended storage conditions for FITC-conjugated LRIT1 antibodies?

FITC-conjugated LRIT1 antibodies should be:

• Stored at 2-8°C for short-term storage

• Protected from light exposure at all times

• Avoided repeated freeze-thaw cycles which can compromise fluorescence intensity

• Maintained in appropriate buffer systems (typically PBS with 0.09% sodium azide and 50% glycerol)

Long-term studies have shown that properly stored FITC-conjugated antibodies can maintain signal intensity for up to 24 months when stored at -20°C .

What is the optimal protocol for immunofluorescence staining using FITC-conjugated LRIT1 antibodies in retinal tissue?

Based on successful protocols for retinal tissue staining:

Materials needed:

• FITC-conjugated LRIT1 antibody

• PBS (pH 7.4)

• Blocking solution (PBS with 10% serum)

• Fixative (4% paraformaldehyde)

• Permeabilization buffer (0.2% Triton X-100 in PBS)

• Mounting medium (aqueous)

Protocol steps:

• Fix tissue sections with 4% paraformaldehyde for 20 minutes

• Wash 3× with PBS, 5 minutes each

• Permeabilize with 0.2% Triton X-100 for 20 minutes

• Block with 10% serum in PBS for 1 hour

• Apply FITC-conjugated LRIT1 antibody (typically 1:300-1:500 dilution) and incubate overnight at 4°C in a dark, humidified chamber

• Wash 3× with PBS, 5 minutes each

• Counterstain nuclei if desired

• Mount with aqueous mounting medium and seal

• Store slides at 4°C in the dark until imaging

How can background autofluorescence be reduced when using FITC-conjugated antibodies in retinal tissue?

Retinal tissue presents challenges with autofluorescence, particularly from lipofuscin and red blood cells. A combinatorial approach has been shown to be most effective:

• Pre-treatment with sodium borohydride: Apply freshly prepared 0.1% sodium borohydride in PBS for 30 minutes to quench fixative-induced autofluorescence

• Crystal violet treatment: Apply 0.05% crystal violet for 5 minutes followed by thorough washing

• Sudan Black B (SBB) application: After antibody staining, treat sections with 0.1% SBB in 70% ethanol for 20 minutes

• Sequential application: The combination of sodium borohydride → crystal violet → SBB in this specific order provides optimal quenching of background while preserving specific FITC signals

This combined approach has been shown to completely eliminate background autofluorescence while maintaining specific LRIT1 immunoreactivity.

How can LRIT1-FITC antibodies be used to study synaptic architecture in the retina?

FITC-conjugated LRIT1 antibodies can be used to investigate synaptic architecture through co-localization studies:

Experimental approach:

• Perform multi-channel immunofluorescence using:

  • LRIT1-FITC antibody (green channel)

  • Photoreceptor synaptic ribbon marker Ctbp2 (different fluorophore)

  • Synaptic cleft marker Pikachurin (different fluorophore)

• Conduct high-resolution confocal microscopy to visualize:

  • LRIT1 localization between horseshoe-like Ctbp2-positive synaptic ribbons

  • Proximity to synaptic clefts stained with Pikachurin

  • Distribution patterns in rod versus cone photoreceptor terminals

• Perform line-scan intensity analysis to quantify the partial overlap of LRIT1 with mGluR6, consistent with its presence in the synaptic cleft

This approach has revealed that LRIT1 signals are observed close to photoreceptor terminals between the synaptic ribbons and synaptic clefts in both rod and cone photoreceptor axon terminals in the OPL.

How does LRIT1 expression change in response to alterations in retinal signaling pathways?

Research has shown remarkable regulation of LRIT1 expression in response to signaling changes:

These findings indicate that LRIT1 expression and synaptic accumulation is inversely dependent on neurotransmitter release orchestrated by the CaV1.4 complex, suggesting a compensatory mechanism in response to synaptic dysfunction .

What are the molecular interactions of LRIT1 that can be studied using co-immunoprecipitation with FITC-labeled antibodies?

LRIT1 engages in several protein-protein interactions that can be investigated:

• LRIT1-mGluR6 interaction:

  • LRIT1 forms a complex with mGluR6 in native retinas

  • This interaction can be confirmed by co-immunoprecipitation followed by western blotting

• LRIT1 homodimerization and heterodimerization:

  • LRIT1 interacts with itself (homodimer)

  • LRIT1 forms heterodimers with LRIT2

  • Minimal interaction occurs with LRIT3

• LRIT1-Frmpd2 interaction:

  • LRIT1 interacts with Frmpd2 through its intracellular domain

  • Specifically, the third PDZ domain (PDZ3) of Frmpd2 interacts with the LRIT1 C-terminal region

For co-IP protocols using FITC-conjugated antibodies, researchers should be aware that the FITC conjugation could potentially affect binding properties, so validation against unconjugated antibodies is recommended.

How can researchers verify the specificity of LRIT1-FITC antibodies?

Multiple validation approaches should be employed:

• Western blot validation:

  • Use cell/tissue lysates from LRIT1 knockout models as negative controls

  • Verify a single band at approximately the predicted molecular weight

  • Compare membrane fraction (should contain LRIT1) with cytosolic fraction (should not contain LRIT1)

• Immunohistochemical validation:

  • Compare staining pattern in wild-type versus LRIT1 knockout tissues

  • Verify absence of staining in the outer plexiform layer in knockout samples

  • Cross-validate with unconjugated antibodies against different epitopes

• Blocking peptide competition:

  • Pre-incubate the FITC-conjugated antibody with excess immunizing peptide

  • Verify elimination of specific staining in this competition assay

What are common pitfalls when working with FITC-conjugated antibodies in retinal tissue?

Several challenges should be anticipated:

• Photobleaching issues:

  • FITC is relatively prone to photobleaching compared to other fluorophores

  • Minimize exposure to excitation light during microscopy

  • Consider anti-fade mounting media containing agents like ProLong Gold

• Tissue autofluorescence:

  • Retinal tissue contains endogenous fluorescent molecules in the same spectrum as FITC

  • Implement autofluorescence quenching methods (see question 2.3)

  • Consider spectral unmixing during image acquisition

• Non-specific binding:

  • FITC-conjugated antibodies may exhibit non-specific binding to highly cationic structures

  • Include appropriate blocking with normal serum (10%) and BSA (1-3%)

  • Consider adding 0.1% Tween-20 to wash buffers to reduce non-specific interactions

How can researchers quantify LRIT1 expression levels using FITC-conjugated antibodies?

For accurate quantification:

• Flow cytometry approach:

  • Dissociate retinal cells using gentle enzymatic digestion

  • Perform intracellular staining with LRIT1-FITC antibody

  • Set appropriate gating based on negative controls

  • Use median fluorescence intensity (MFI) for quantitative comparison between samples

• Fluorescence microscopy quantification:

  • Standardize all image acquisition parameters (exposure time, gain, etc.)

  • Include internal calibration standards in each imaging session

  • Analyze images using software like ImageJ for:

    • Integrated density measurements

    • Background-subtracted mean fluorescence intensity

    • Colocalization coefficients with synaptic markers

• Controls for quantification:

  • Include isotype control antibodies conjugated to FITC

  • Use tissues from knockout animals as negative controls

  • Include samples with known expression levels as reference standards

How can LRIT1-FITC antibodies be used to investigate changes in retinal adaptation mechanisms?

Based on research showing LRIT1's role in adaptation:

Experimental design approach:

• Subject model animals (wild-type and LRIT1-knockout) to varying light adaptation protocols

• Prepare retinal sections at specific timepoints during adaptation

• Perform immunostaining with LRIT1-FITC and markers for ON-bipolar cells

• Analyze:

  • Changes in LRIT1 localization during adaptation

  • Alterations in synaptic architecture

  • Correlations with electrophysiological measurements

  • Differences between wild-type and heterozygous animals

This type of investigation has revealed that LRIT1 knockout mice show increased sensitivity of cone synaptic signaling while impairing adaptation to background light, suggesting LRIT1's role in scaling synaptic communication.

What methodological considerations are important when performing multi-color immunofluorescence including LRIT1-FITC antibodies?

For optimal multi-color imaging:

• Fluorophore selection:

  • Pair FITC (excitation/emission: 495nm/524nm) with spectrally distinct fluorophores

  • Recommended combinations: FITC + Cy3/TRITC + Cy5 or DAPI + FITC + TRITC

  • Avoid fluorophores with significant spectral overlap to minimize bleed-through

• Antibody compatibility:

  • When combining multiple primary antibodies, select those raised in different host species

  • For same-species antibodies, consider sequential staining with blocking steps

  • Validate that antibody binding sites don't interfere with each other

• Staining procedure:

  • Apply FITC-conjugated antibodies last in the sequence when possible

  • Extend washing steps to ensure complete removal of unbound antibodies

  • Consider spectral unmixing during image acquisition for closely overlapping fluorophores

How can researchers design experiments to study LRIT1 molecular interactions in photoreceptor synapses?

Based on successful approaches in the literature:

Recommended experimental workflow:

• Primary characterization:

  • Use LRIT1-FITC antibodies to map expression patterns in retinal sections

  • Co-stain with markers for pre- and post-synaptic structures

• Potential interaction partners identification:

  • Perform immunoprecipitation using anti-LRIT1 antibodies

  • Analyze precipitates using mass spectrometry

  • Validate interactions with co-IP and western blotting

• Functional validation:

  • Compare wild-type and LRIT1 knockout mouse models

  • Analyze synaptic transmission using electrophysiology

  • Correlate physiological findings with protein localization

  • Consider rescue experiments with wild-type or mutant LRIT1

This comprehensive approach has successfully identified several LRIT1 interaction partners and established its role in modulating adaptive changes in synaptic communication of cone photoreceptors.