LAMA4 Antibody, FITC conjugated

What is LAMA4 and why is it important for immunofluorescence studies?

Laminin subunit alpha-4 (LAMA4) is an extracellular matrix glycoprotein that mediates cell attachment, migration, and organization during tissue development. LAMA4 is particularly significant because:

• It functions as a key component of basement membranes by binding to cells via high-affinity receptors

• It demonstrates tissue-specific expression patterns with strong presence in heart, lung, ovary, intestines, placenta, and liver

• It shows differential expression in fetal tissues, particularly in lung and kidney, and in mesenchymal cells such as smooth muscle and dermis

• It plays crucial roles in directing cell migration and organization through microspike and filopodia formation

For immunofluorescence studies, FITC-conjugated LAMA4 antibodies provide direct visualization without requiring secondary antibodies, offering enhanced specificity for examining LAMA4 distribution in tissues and cultured cells.

What are the typical specifications of commercially available LAMA4-FITC antibodies?

Common specifications for LAMA4-FITC antibodies include:

Most LAMA4-FITC antibodies are purified by antigen affinity chromatography and supplied in buffer containing glycerol as a preservative .

How do I determine the optimal antibody concentration for LAMA4-FITC in my experiments?

Determining optimal concentration requires systematic titration:

• Begin with manufacturer's recommended range (typically 1-10 μg/mL for immunofluorescence)

• Perform a dilution series experiment using:

  • Positive control tissue known to express LAMA4 (e.g., placenta, heart tissue)

  • Negative control tissue with minimal LAMA4 expression (e.g., specific regions of brain tissue)

• Include isotype controls at equivalent concentrations to assess non-specific binding

• Establish signal-to-noise ratio at each concentration by comparing:

  • Mean fluorescence intensity in positive regions

  • Background fluorescence in negative regions

Optimal dilutions should provide clear visualization of expected staining patterns (e.g., basement membrane localization, extracellular matrix structures) with minimal background. For most applications, signal-to-noise ratios above 3:1 are considered acceptable for quantitative analysis.

What are the recommended protocols for tissue sample preparation when using LAMA4-FITC antibodies?

For optimal results with LAMA4-FITC antibodies, tissue preparation is critical:

For Paraffin-Embedded Tissues:

• Fix tissues in 4% formaldehyde/paraformaldehyde for 24 hours

• Process and embed in paraffin following standard protocols

• Section tissues at 3-5 μm thickness

• Perform heat-induced epitope retrieval using alkaline buffer (pH 9.0) for optimal LAMA4 antigen recovery

• Block endogenous fluorescence with 0.1% sodium borohydride solution

• Apply LAMA4-FITC antibody at predetermined concentration (typically 1:100 to 1:200 dilution)

• Counterstain nuclei with DAPI or Hoechst 33342

• Mount with anti-fade mounting medium

For Frozen Sections and Cultured Cells:

• Fix in 3.7% formaldehyde for 10-15 minutes at room temperature

• Permeabilize if necessary (0.1-0.2% Triton X-100 for 5-10 minutes)

• Block with 1-5% BSA or serum from the same species as secondary antibody

• Incubate with LAMA4-FITC antibody at 4°C overnight or 1-2 hours at room temperature

• Wash extensively with PBS

• Counterstain and mount as above

How can I distinguish specific from non-specific staining when using LAMA4-FITC antibodies?

To ensure staining specificity:

• Include proper controls:

  • Isotype control antibody (same species, isotype, and conjugate as LAMA4-FITC)

  • Secondary antibody-only control (for detecting autofluorescence)

  • Known positive and negative tissue controls

  • Blocking peptide competition (pre-incubation of antibody with immunizing peptide)

• Validate staining pattern:

  • LAMA4 should primarily localize to basement membranes and extracellular matrix

  • Strong expression patterns should correlate with known expression in heart, lung, ovary, intestines, placenta, and liver

  • Staining should be reduced in tissues known to have low expression (skeletal muscle, brain)

• Cross-validate with alternative methods:

  • Confirm results using antibodies targeting different LAMA4 epitopes

  • Compare with RNA expression data (e.g., from publicly available databases)

  • Use LAMA4 knockout or knockdown models as negative controls when possible

What quantification methods are most appropriate for LAMA4-FITC immunofluorescence experiments?

For rigorous quantification of LAMA4 staining:

• Image acquisition standardization:

  • Use consistent exposure settings across all experimental groups

  • Acquire images below saturation

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

• Analysis approaches by experiment type:

  For tissue sections:

  • Measure mean fluorescence intensity (MFI) in regions of interest

  • Quantify LAMA4-positive area as percentage of total tissue area

  • Measure basement membrane thickness/continuity using line profile analysis

  For cell cultures:

  • Count cells with LAMA4 positivity above threshold

  • Measure MFI per cell

  • Analyze colocalization with other markers (e.g., integrin receptors)

• Software options:

  • ImageJ/FIJI with appropriate plugins

  • CellProfiler for automated analysis

  • Commercial software packages (Imaris, Columbus, etc.)

• Statistical analysis:

  • Compare MFI using appropriate statistical tests (t-test, ANOVA)

  • Account for potential confounders (tissue thickness variations, autofluorescence)

  • Use normalization to internal controls when comparing across experiments

How can LAMA4-FITC antibodies be used to study cell migration and cellular protrusions?

LAMA4 plays a crucial role in directed cell migration through regulation of cellular protrusions. FITC-conjugated LAMA4 antibodies enable detailed visualization of these processes:

• Live-cell imaging applications:

  • Monitor LAMA4 deposition during cell migration in real-time

  • Track dynamic changes in LAMA4-rich microenvironments

  • Observe formation of microspikes and filopodia in response to LAMA4

• Experimental approaches:

  • Perform scratch assays on LAMA4-coated surfaces with FITC-stained cells

  • Use microfluidic chambers to establish LAMA4 gradients and track cell movement

  • Combine with cytoskeletal markers (F-actin) to correlate LAMA4 with protrusion formation

• Quantitative analysis:

  • Measure directionality and velocity of cell migration

  • Quantify number and length of cellular protrusions

  • Assess cluster formation rates in relation to LAMA4 expression

Research findings show that LAMA4 blockade significantly reduces microspike formation and affects cell surface morphology, suggesting LAMA4 is involved in directed cell motility rather than general migration capacity .

What are the recommended protocols for multiplexed immunofluorescence including LAMA4-FITC antibodies?

For multiplexed staining:

• Panel design considerations:

  • Choose fluorophores with minimal spectral overlap

  • Pair FITC (excitation 499nm/emission 515nm) with compatible fluorophores:

    • DAPI for nuclei (excitation 358nm/emission 461nm)

    • Cy3/TRITC for second target (excitation ~550nm/emission ~570nm)

    • Cy5/AlexaFluor 647 for third target (excitation ~650nm/emission ~670nm)

• Sequential staining protocol:

  • Begin with the weakest signal antibody

  • Include appropriate blocking steps between antibodies

  • Consider order of antibody application based on host species

• Example protocol for LAMA4 with immune cell markers:

  • Fix and permeabilize tissue/cells as described earlier

  • Block with 5% normal serum/1% BSA for 1 hour

  • Apply LAMA4-FITC antibody (1:100) overnight at 4°C

  • Wash extensively with PBS (3-5 times, 5 minutes each)

  • Apply additional primary antibodies (e.g., CD11c for dendritic cells)

  • Add compatible fluorophore-conjugated secondary antibodies

  • Counterstain nuclei with DAPI

  • Mount with anti-fade medium

• Controls specific to multiplexed staining:

  • Single-stain controls for compensation/spectral unmixing

  • Fluorescence-minus-one (FMO) controls

  • Absorption controls if using multiple antibodies from same species

How can LAMA4-FITC antibodies be used to investigate the role of LAMA4 in lymph node conduit structures?

LAMA4 has been shown to be critical for lymph node conduit integrity and immune function. FITC-conjugated LAMA4 antibodies can be used to study these specialized structures:

• Experimental design for conduit visualization:

  • Prepare thin (5-10 μm) cryosections of lymph nodes

  • Use LAMA4-FITC antibodies (1:100-1:200) to visualize conduit structure

  • Co-stain with markers for fibroblastic reticular cells (FRCs) such as PDPN or ER-TR7

  • Include markers for immune cells (T cells, B cells, dendritic cells)

• Functional analysis of conduits:

  • Inject fluorescent dextran tracer molecules (10-70 kDa) to assess conduit flow

  • Monitor antigen transport through LAMA4-rich conduits

  • Quantify colocalization of antigens with LAMA4-positive structures

  • Measure immune cell associations with LAMA4-positive conduits

• Applications in LAMA4-deficient models:

  • Compare conduit structure between wild-type and LAMA4-knockout tissues

  • Assess restoration of conduit function after FRC transplantation

  • Analyze Treg and chemokine distribution in relation to LAMA4 expression

  • Evaluate immune response alterations in LAMA4-deficient conditions

Recent research demonstrates that FRC-derived LAMA4 is essential for maintaining conduit structures in lymph nodes, with LAMA4 deficiency leading to impaired antigen transport and immune cell distribution .

How can I resolve weak or non-specific staining when using LAMA4-FITC antibodies?

Common issues and solutions:

For weak or absent staining:

• Epitope masking: Optimize antigen retrieval methods (try different pH buffers, increased retrieval time)

• Insufficient antibody concentration: Increase antibody concentration in a stepwise manner

• Antibody incompatibility with fixation: Test different fixation methods (paraformaldehyde, methanol)

• Degraded antibody: Use fresh aliquots and avoid repeated freeze-thaw cycles

• Low LAMA4 expression: Confirm expression level through other methods (WB, qPCR)

For high background or non-specific staining:

• Insufficient blocking: Increase blocking time/concentration (5-10% normal serum)

• Autofluorescence: Pretreat with sodium borohydride (0.1% for 10 minutes) or use commercially available autofluorescence quenching reagents

• Non-specific binding: Include 0.1-0.3% Triton X-100 in antibody diluent

• Overfixation: Reduce fixation time or concentration

• Tissue-specific issues: For certain tissues (liver, kidney), include additional blocking steps with avidin/biotin blocking kit

What are the latest research findings regarding LAMA4's role in cancer progression that can be studied using FITC-conjugated antibodies?

Recent studies reveal LAMA4's significance in cancer research, which can be further explored using FITC-conjugated antibodies:

• LAMA4 in tumor microenvironment:

  • LAMA4 expression is linked to prognosis in gastric cancer (GC)

  • LAMA4 expression correlates with immune cell infiltration, including macrophages, CD4+ T cells, and dendritic cells

  • LAMA4 shows associations with markers of M2 and tumor-associated macrophages (TAMs)

• Research applications with LAMA4-FITC:

  • Quantify LAMA4 expression in primary tumors versus metastatic sites

  • Analyze spatial relationships between LAMA4-positive structures and infiltrating immune cells

  • Track LAMA4 expression changes during epithelial-mesenchymal transition

  • Correlate LAMA4 patterns with clinical outcomes and treatment responses

• Methodological approaches:

  • Multiplex staining with LAMA4-FITC and immune cell markers

  • Co-localization analysis with basement membrane components

  • Comparison of LAMA4 expression in tumor versus adjacent normal tissue

  • Quantitative assessment of LAMA4 in relation to tumor invasion patterns

How can flow cytometry be optimized when using LAMA4-FITC antibodies for detecting cell surface or intracellular LAMA4?

Optimizing flow cytometry with LAMA4-FITC antibodies:

• Sample preparation considerations:

  • For cell surface LAMA4: Use gentle enzymatic dissociation methods to preserve extracellular epitopes

  • For intracellular LAMA4: Fix cells with 2-4% paraformaldehyde followed by permeabilization with 0.1-0.5% saponin or 0.1% Triton X-100

• Staining protocol optimization:

  • Cell concentration: 1-5 × 10^6 cells/mL

  • LAMA4-FITC concentration: Typically 0.5-5 μg/mL (optimize through titration)

  • Incubation conditions: 30-60 minutes at 4°C (surface) or room temperature (intracellular)

  • Buffer composition: PBS with 1-2% BSA and 0.1% sodium azide

• Flow cytometer settings:

  • Excitation: 488 nm laser

  • Emission filter: 530/30 nm bandpass

  • Compensation: Set up using FITC single-stained controls

  • Voltage adjustment: Optimize to place negative population in first decade of log scale

• Controls and validation:

  • Unstained cells for autofluorescence assessment

  • Isotype-FITC control for non-specific binding

  • FMO controls for accurate gating in multicolor panels

  • Blocking peptide competition to confirm specificity

• Analysis considerations:

  • Gate on viable cells (using viability dye)

  • Analyze LAMA4 expression as mean/median fluorescence intensity

  • For heterogeneous populations, consider subset analysis based on LAMA4 expression levels

• Co-expression analysis:

  • Combine with markers for specific cell types (e.g., endothelial cells, fibroblasts)

  • Analyze LAMA4 expression in relation to activation or differentiation markers

  • Correlate with functional properties (e.g., migration capacity, proliferation)