LIMS1 Antibody, FITC conjugated

What is LIMS1 and why is it relevant for research?

LIMS1, also known as PINCH1, is a widely expressed protein in mammalian cells that plays crucial roles in cytoskeletal organization and cell-cell junctions. It contains LIM and senescent cell antigen-like domains that facilitate protein-protein interactions . LIMS1 is particularly important for research because of its involvement in several physiological and pathological processes. Recent studies have identified LIMS1 as a potential target for pathogenic autoimmunity in conditions such as pathologic myopia and kidney allograft rejection . Its highest expression has been observed in retinal pigment epithelial (RPE) cells, suggesting its critical role in maintaining the outer blood-retinal barrier .

What are the key specifications to consider when selecting a LIMS1 antibody?

When selecting a LIMS1 antibody for research, consider these critical parameters:

Antibodies targeting different epitopes may yield different results depending on protein conformation and accessibility in your experimental system.

What advantages does FITC conjugation offer for LIMS1 detection?

FITC conjugation provides several methodological advantages for LIMS1 detection:

• Direct visualization without secondary antibodies, reducing background and cross-reactivity issues

• Simplified protocols with fewer washing steps and incubation periods

• Compatibility with standardized filter sets on most fluorescence microscopes and flow cytometers

• Strong fluorescence signal (excitation ~495 nm, emission ~519 nm) that pairs well with other fluorophores in multi-color analyses

• Elimination of secondary antibody cross-reactivity concerns in multi-species co-staining experiments

• Quantitative capabilities for flow cytometry and digital image analysis of expression levels

Which experimental approaches benefit most from FITC-conjugated LIMS1 antibodies?

FITC-conjugated LIMS1 antibodies are particularly valuable for:

• Immunocytochemistry and immunohistochemistry to visualize LIMS1 distribution in tissues and cells, especially in RPE cells where LIMS1 is highly expressed

• Flow cytometry for quantifying LIMS1 expression across cell populations

• Live-cell imaging applications where minimal processing is preferred

• Tissue sections where penetration of multiple antibodies might be challenging

• Co-localization studies with proteins detected by antibodies from the same host species

How can I validate the specificity of a FITC-conjugated LIMS1 antibody?

Thorough validation is essential for ensuring reliable results with FITC-conjugated LIMS1 antibodies:

• Western blot validation: First test an unconjugated version of the same antibody clone on cell lysates known to express LIMS1 (e.g., ARPE-19 cells) to confirm correct molecular weight detection (~37 kDa)

• Positive and negative controls: Include tissues/cells with known high LIMS1 expression (e.g., RPE cells) and those with minimal expression

• Peptide competition assay: Pre-incubate antibody with excess purified LIMS1 protein (Ala68-Cys303) to confirm signal reduction in immunostaining

• siRNA knockdown: Perform parallel staining on LIMS1-knockdown cells to demonstrate specificity through signal reduction

• Orthogonal detection methods: Compare results with antibodies targeting different LIMS1 epitopes or with mRNA expression by in situ hybridization

• Immunoprecipitation followed by mass spectrometry: Confirm that the antibody pulls down LIMS1 protein specifically

Proper validation protocols increase confidence in experimental results and support reproducibility across studies.

What is the optimal protocol for conjugating LIMS1 antibodies with FITC in-house?

For researchers wanting to perform their own FITC conjugation:

• Recommended method: Use a Lightning-Link® FITC Conjugation Kit for rapid and efficient labeling with minimal hands-on time (approximately 30 seconds)

• Protocol overview:

  • Prepare antibody in appropriate buffer (typically PBS) at 1-4 mg/ml

  • Add LL-Modifier reagent to the antibody

  • Transfer to the lyophilized FITC mix

  • Incubate for 3-4 hours at room temperature

  • Add LL-Quencher if the conjugate will not be used immediately

• Advantages: 100% antibody recovery, conjugation in less than 4 hours, and compatibility with small quantities of antibody

• Quality control: After conjugation, verify success by spectrophotometric analysis (absorption at 495 nm for FITC and 280 nm for protein)

• Storage: Store conjugated antibody at 4°C protected from light for short-term use, or aliquot and freeze at -20°C for long-term storage

Studies by Lim et al. have successfully used this approach for conjugating antibodies for immunohistochemistry in neural tissue research .

How does LIMS1 expression pattern correlate with pathological conditions?

Recent research has revealed significant correlations between LIMS1 expression/autoimmunity and pathological conditions:

• Pathologic myopia: Anti-LIMS1 autoantibodies have been identified as potential biomarkers specifically in pathologic myopia characterized by myopic macular degeneration (MMD), rather than simple high myopia

• Autoimmune profile: In pathologic myopia, anti-LIMS1 autoantibodies primarily belong to IgG1/IgG2/IgG3 subclasses, suggesting a specific immune response

• Mechanism of action: These autoantibodies appear to disrupt the blood-retinal barrier by interfering with cytoskeletal organization and cellular tight junctions in RPE cells

• Inflammatory response: Anti-LIMS1 autoantibodies induce upregulation of inflammatory markers such as ICAM-1 and IL-9, creating a pro-inflammatory state in RPE cells

• Kidney allograft rejection: LIMS1 has been implicated in this autoimmune disorder, resulting from hypoxia-induced upregulation of the LIMS1 antigen in human kidney cells

These findings suggest that FITC-conjugated LIMS1 antibodies may be valuable tools in investigating the role of this protein in various disease processes.

What optimization steps improve FITC-LIMS1 antibody performance in confocal microscopy?

For optimal confocal microscopy results with FITC-conjugated LIMS1 antibodies:

• Fixation optimization:

  • For cytoskeletal structures: 4% paraformaldehyde for 15 minutes at room temperature

  • For membrane structures: Methanol fixation at -20°C for 10 minutes

• Signal preservation:

  • Minimize photobleaching by using antifade mounting media

  • Store slides at 4°C in darkness

  • Image samples promptly after preparation

• Background reduction:

  • Include a 30-minute blocking step with 5% serum from the same species as the secondary antibody

  • Add 0.1-0.3% Triton X-100 for effective permeabilization

  • Include 0.05% Tween-20 in wash buffers

• Imaging parameters:

  • Use appropriate excitation wavelength (488 nm laser)

  • Set emission collection around 515-530 nm

  • Optimize pinhole settings (1 Airy unit recommended)

  • Adjust detector gain to avoid saturation while maximizing signal

• Counterstaining compatibility:

  • DAPI works well for nuclear counterstaining with FITC

  • For co-localization studies with cytoskeletal elements, consider phalloidin-rhodamine for F-actin

These optimizations are particularly important when studying LIMS1's role in cytoskeletal organization and cellular junctions.

How can I use FITC-conjugated LIMS1 antibodies to investigate blood-retinal barrier function?

Based on recent findings linking LIMS1 to blood-retinal barrier integrity, researchers can implement these methodological approaches:

• Immunolocalization in retinal cross-sections:

  • Process human or animal eye tissue with standard fixation

  • Perform immunostaining with FITC-conjugated LIMS1 antibody

  • Counterstain with markers for tight junction proteins (ZO-1, claudin-5)

  • Analyze co-localization at the RPE layer using confocal microscopy

• In vitro barrier function assays:

  • Culture ARPE-19 cells on transwell inserts until confluent

  • Assess barrier integrity by transepithelial electrical resistance (TEER) measurements

  • Perform FITC-dextran permeability assays before and after treatment with potential barrier disruptors

  • Correlate changes with LIMS1 localization using the FITC-conjugated antibody

• Manipulation of LIMS1 expression:

  • Use siRNA knockdown or CRISPR-Cas9 to modify LIMS1 expression in RPE cells

  • Monitor effects on tight junction formation and barrier function

  • Confirm protein reduction using FITC-conjugated LIMS1 antibodies in flow cytometry

• Autoantibody effect studies:

  • Isolate IgG from patients with pathologic myopia

  • Treat RPE cell monolayers with these antibodies

  • Visualize changes in LIMS1 distribution and barrier integrity

  • Deplete anti-LIMS1 autoantibodies to confirm specificity of effects

This approach has revealed that anti-LIMS1 autoantibodies can disrupt RPE barrier function, potentially contributing to macular degeneration in pathologic myopia .

What protocols enable effective dual immunofluorescence with FITC-conjugated LIMS1 antibodies?

For successful multi-color immunofluorescence incorporating FITC-conjugated LIMS1 antibodies:

• Companion fluorophore selection:

  • Choose fluorophores with minimal spectral overlap with FITC (e.g., TRITC, Cy5)

  • For triple labeling, consider DAPI (blue), FITC-LIMS1 (green), and Cy5 (far-red)

• Sequential staining protocol:

  • Fix cells/tissues appropriately (4% PFA for 15 minutes)

  • Block with 5% normal serum + 0.3% Triton X-100 for 1 hour

  • Apply FITC-conjugated LIMS1 antibody first (typically 1:100-1:200 dilution)

  • Wash extensively (3-5 times with PBS-T)

  • Apply unconjugated primary antibody for second target

  • Wash again thoroughly

  • Apply appropriate secondary antibody with compatible fluorophore

  • Perform final washes and mount with antifade medium

• Controls for multi-color experiments:

  • Single-color controls to assess bleed-through

  • Secondary-only control to evaluate background

  • Isotype control to confirm specificity

• Example multi-label combination in RPE research:

  • FITC-LIMS1 antibody

  • Anti-ZO-1 (tight junction marker) with Cy5-conjugated secondary

  • DAPI for nuclear counterstaining

This approach has been successfully employed to study traumatic nerve injury and hypoxia, using FITC-conjugated antibodies alongside other markers .

How should FITC-conjugated LIMS1 antibodies be used in flow cytometry?

For optimal flow cytometry results with FITC-conjugated LIMS1 antibodies:

• Cell preparation:

  • Harvest cells using enzymatic methods (trypsin/EDTA) or mechanical dissociation

  • Fix with 2-4% paraformaldehyde for 10-15 minutes

  • Permeabilize with 0.1% saponin or 0.1% Triton X-100 if detecting intracellular LIMS1

• Staining protocol:

  • Block with 2% BSA in PBS for 30 minutes

  • Incubate with FITC-conjugated LIMS1 antibody (typically 1 μg per 10^6 cells)

  • Incubate for 45-60 minutes at room temperature in darkness

  • Wash 3 times with PBS containing 0.1% BSA

  • Resuspend in appropriate buffer for analysis

• Controls and optimization:

  • Include unstained cells for autofluorescence assessment

  • Use isotype control (FITC-conjugated non-specific IgG) at same concentration

  • Titrate antibody to determine optimal concentration

  • Include compensation controls if performing multi-color analysis

• Analysis considerations:

  • Gate on viable cells using appropriate exclusion dye

  • Analyze FITC signal on FL1 channel (typically 530/30 nm bandpass filter)

  • Present data as histogram or dot plot against other parameters

  • Calculate percent positive cells and mean fluorescence intensity

This approach has been effectively used with FITC-conjugated antibodies to examine membrane protein expression in lung, breast, and colorectal cancer cell lines .

What factors should be considered when interpreting LIMS1 immunohistochemistry results?

Critical considerations for accurate interpretation of LIMS1 immunohistochemistry:

• Cellular localization interpretation:

  • LIMS1 typically localizes to focal adhesions and cell-cell junctions

  • Aberrant cytoplasmic or nuclear localization may indicate pathology

  • In RPE cells, LIMS1 is strongly expressed and involved in maintaining barrier function

• Expression pattern variations:

  • Higher expression is normal in certain tissues (e.g., RPE cells)

  • Consider regional variation within tissues

  • Compare pathological samples to appropriate normal controls

• Quantitative assessment approaches:

  • Use digital image analysis for objective quantification

  • Measure both intensity and distribution patterns

  • Consider area fraction of positive staining

• Technical artifacts vs. true signal:

  • Edge artifacts in tissue sections can be misinterpreted

  • Autofluorescence, particularly in eye tissues, may confound results

  • Fixation differences can affect epitope accessibility and signal strength

• Validation through multiple approaches:

  • Confirm key findings with alternative techniques (Western blot, qPCR)

  • Use in situ hybridization to correlate protein with mRNA expression

  • Consider co-localization with known interaction partners

These considerations are particularly important given LIMS1's involvement in pathologic myopia and its potential role as a biomarker .

How can I resolve weak or inconsistent signal when using FITC-conjugated LIMS1 antibodies?

When encountering signal issues with FITC-conjugated LIMS1 antibodies:

• Antibody-related troubleshooting:

  • Check antibody viability (FITC is sensitive to light and pH)

  • Titrate antibody concentration (try 2-5X higher concentration)

  • Consider alternative clones targeting different epitopes of LIMS1

  • Verify antibody reactivity matches species being studied

• Sample preparation optimization:

  • Test different fixation methods (4% PFA, methanol, or acetone)

  • Optimize permeabilization (vary Triton X-100 concentration from 0.1-0.5%)

  • Try antigen retrieval (citrate buffer pH 6.0, 95°C for 20 minutes)

  • Extend primary antibody incubation time (overnight at 4°C)

• Signal amplification options:

  • Use anti-FITC antibodies conjugated to brighter fluorophores

  • Implement tyramide signal amplification system

  • Consider biotin-streptavidin amplification

• Technical adjustments:

  • Increase exposure time or detector gain during imaging

  • Reduce washing stringency (use gentler buffers, shorter wash times)

  • Optimize mounting media (use one specifically designed to preserve FITC)

  • Protect from photobleaching during all steps

• Experimental controls:

  • Include positive control samples known to express high levels of LIMS1 (e.g., RPE cells)

  • Process all samples in parallel to identify technical variables

What are the best practices for quantifying LIMS1 expression using immunofluorescence?

For reliable quantification of LIMS1 expression in immunofluorescence studies:

• Image acquisition standardization:

  • Use identical microscope settings for all experimental groups

  • Capture multiple random fields per sample (minimum 5-10)

  • Include exposure settings in metadata

  • Collect z-stacks for three-dimensional samples

• Quantification methodologies:

  • Mean fluorescence intensity (MFI) within defined regions of interest

  • Integrated density (area × mean intensity)

  • Colocalization coefficients when performing dual staining

  • Cell-by-cell analysis for population distributions

• Software tools:

  • ImageJ/FIJI with appropriate plugins

  • CellProfiler for automated multi-parameter analysis

  • Commercial options: MetaMorph, Imaris, or ZEN software

• Normalization approaches:

  • Use internal controls (unchanging housekeeping proteins)

  • Include fluorescent beads as intensity standards

  • Express results as fold change relative to control samples

• Statistical analysis:

  • Determine appropriate statistical tests based on data distribution

  • Consider both biological and technical replicates

  • Present results with suitable error bars (SEM or SD)

  • Include sample size and p-values

This quantitative approach has been valuable in studies examining the role of LIMS1 in pathologic myopia and other conditions .

How can I design experiments to investigate LIMS1's role in cytoskeletal organization?

To investigate LIMS1's role in cytoskeletal organization:

• Co-localization studies:

  • Perform dual immunofluorescence with FITC-conjugated LIMS1 antibody and markers for:

    • Actin filaments (phalloidin-TRITC)

    • Focal adhesions (vinculin, paxillin)

    • Cell-cell junctions (β-catenin, E-cadherin)

  • Analyze using confocal microscopy and quantitative colocalization metrics

• Functional perturbation approaches:

  • siRNA knockdown of LIMS1 expression

  • CRISPR-Cas9 gene editing to create LIMS1 mutants

  • Overexpression of wild-type or mutant LIMS1

  • Treatment with cytoskeletal disruptors (cytochalasin D, nocodazole)

• Protein-protein interaction analysis:

  • Co-immunoprecipitation with LIMS1 antibodies followed by Western blotting for interaction partners

  • Proximity ligation assay to visualize protein interactions in situ

  • FRET or BRET analysis for dynamic interactions

• Live-cell imaging:

  • Express LIMS1-GFP fusion proteins to track dynamics

  • Perform fluorescence recovery after photobleaching (FRAP) to assess mobility

  • Use photoactivatable or photoconvertible LIMS1 constructs to track protein movement

• Mechanical stress response:

  • Apply mechanical strain to cell monolayers

  • Observe LIMS1 redistribution using FITC-conjugated antibodies

  • Correlate with changes in cytoskeletal architecture and cell-cell junctions

These approaches can provide insights into LIMS1's role in maintaining RPE barrier function and cytoskeletal organization, relevant to pathologic myopia pathogenesis .

What strategies can detect autoantibodies against LIMS1 in patient samples?

Based on recent research on anti-LIMS1 autoantibodies in pathologic myopia, these methodological approaches are recommended:

• ELISA-based detection:

  • Coat plates with purified LIMS1 protein (50 ng/well)

  • Incubate with patient serum (typically 1:100 dilution)

  • Detect using HRP-conjugated anti-human IgG

  • Include controls from healthy individuals

  • Normalize results to mean values from emmetropia group

• Immunoglobulin class and subclass profiling:

  • Perform parallel ELISAs with isotype-specific secondary antibodies:

    • Anti-human IgG, IgM, IgA, and IgE

    • Anti-human IgG1, IgG2, IgG3, and IgG4

  • Quantify relative levels of each antibody type

• Western blot confirmation:

  • Separate LIMS1 protein by SDS-PAGE

  • Transfer to PVDF membrane

  • Incubate with patient serum followed by HRP-conjugated anti-human IgG

  • Compare band intensity to positive and negative controls

• Functional assays:

  • Purify IgG from patient serum

  • Test effects on ARPE-19 cells in culture

  • Monitor barrier function via TEER measurements

  • Visualize cytoskeletal changes with appropriate stains

  • Include control conditions with anti-LIMS1 depleted IgG

• Immunofluorescence on transfected cells:

  • Express LIMS1 in suitable cell lines

  • Incubate with patient serum

  • Detect with fluorophore-conjugated anti-human IgG

  • Compare to known positive and negative controls

These methods have successfully identified anti-LIMS1 autoantibodies as potential biomarkers for pathologic myopia .

What are the emerging applications of FITC-conjugated LIMS1 antibodies in clinical research?

FITC-conjugated LIMS1 antibodies show promising applications in clinical research:

• Biomarker development: The identification of LIMS1 autoantibodies in pathologic myopia suggests potential diagnostic applications for detecting and monitoring disease progression

• Mechanistic studies: These tools enable investigation of cytoskeletal and junction disruptions in RPE cells that may contribute to blood-retinal barrier breakdown in various retinal diseases

• Therapeutic target identification: Understanding LIMS1's role in maintaining cellular junctions could lead to novel treatments aimed at preserving barrier function

• Autoimmune disease research: The involvement of LIMS1 in both pathologic myopia and kidney allograft rejection suggests broader implications in autoimmune pathologies

• Drug screening applications: FITC-conjugated LIMS1 antibodies can facilitate high-content screening of compounds that may stabilize LIMS1-dependent cellular junctions