FREM2 Antibody, FITC conjugated

What is FREM2 and why is it important in research?

FREM2 (FRAS1-related extracellular matrix protein 2) is an extracellular matrix protein required for maintaining the integrity of skin epithelium and renal epithelia. It plays a crucial role in epidermal adhesion and is involved in the development of eyelids and the anterior segment of eyeballs . Research on FREM2 is important because it appears to be an essential gene during embryonic development, with its dysfunction potentially linked to developmental abnormalities . Studies using FREM2 antibodies enable researchers to better understand its distribution and function in tissues, providing insights into both normal developmental processes and pathological conditions.

What are the main applications for FREM2 antibody with FITC conjugation?

FREM2 antibody with FITC conjugation is primarily used in fluorescence-based detection methods. The main applications include:

• Enzyme-linked immunosorbent assay (ELISA) for quantitative detection of FREM2 in samples

• Fluorescence microscopy for visualizing FREM2 localization in tissues and cells

• Potential use in flow cytometry for detecting FREM2 in cell populations

• Immunofluorescence studies to examine FREM2 distribution in relation to other cellular components

• Possibly in fluorescence in situ hybridization (FISH) when combined with nucleic acid probes

FITC conjugation provides direct fluorescent visualization without requiring secondary antibodies, streamlining the experimental workflow and potentially reducing background signal in certain applications.

What sample types can be analyzed using FREM2 antibody, FITC conjugated?

FREM2 antibody with FITC conjugation has been validated for use with human samples . The antibody can detect FREM2 in various sample types including:

• Formalin-fixed, paraffin-embedded (FFPE) tissue sections, particularly from human breast and kidney tissues

• Cell culture samples, especially those expressing FREM2

• Potentially in frozen tissue sections, although specific validation may be required

• Protein lysates for techniques such as ELISA

When working with novel sample types, researchers should conduct preliminary validation studies to confirm antibody performance in their specific experimental context.

What are the optimal storage conditions for FREM2 antibody, FITC conjugated?

For optimal performance and longevity, FREM2 antibody with FITC conjugation should be stored according to these guidelines:

• Ship at 4°C to maintain antibody integrity during transport

• Upon receipt, store at -20°C for short-term storage

• For long-term storage, maintain at -80°C to preserve both antibody function and FITC fluorescence

• Avoid repeated freeze-thaw cycles which can degrade both the antibody and the fluorophore

• Store in the dark whenever possible, as FITC is light-sensitive and can photobleach with extended exposure

The antibody is typically supplied in a buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4, which helps maintain stability during storage .

How does FREM2 antibody specificity compare between different mammalian species?

FREM2 antibody specificity varies across mammalian species due to sequence homology differences. While the commercially available antibodies have been validated for human samples , cross-reactivity with mouse models might be expected given that FREM2 is highly conserved functionally across species. The mouse FREM2 protein is approximately 3160 amino acids in length with a large extracellular domain , and researchers investigating FREM2 in mouse models have used specific antibodies for detection in sensory neuron studies .

When planning cross-species studies, researchers should:

• Perform sequence alignment analysis between human and target species FREM2 sequences

• Verify the immunogen sequence used to generate the antibody (typically a synthetic peptide within human FREM2 or recombinant human FREM2 protein fragments)

• Conduct preliminary validation studies using positive and negative controls from the target species

• Consider custom antibody development for species with low homology to human FREM2

What controls should be implemented when using FREM2 antibody, FITC conjugated, in dual-labeling experiments?

Dual-labeling experiments with FREM2 antibody, FITC conjugated, require rigorous controls to ensure reliable results:

• Spectral overlap controls:

  • Single-stained samples for each fluorophore to establish spectral profiles

  • Compensation controls if using flow cytometry

  • Selection of compatible secondary fluorophores that minimize overlap with FITC (excitation ~495 nm, emission ~520 nm)

• Antibody specificity controls:

  • Isotype control (rabbit IgG-FITC) to assess non-specific binding

  • Blocking peptide competition assay using the immunogen peptide

  • Tissues/cells known to be negative for FREM2 expression

• Technical controls:

  • Auto-fluorescence control (unstained sample)

  • FITC-only control without primary antibody

  • Secondary antibody-only control if using additional antibodies

• Cross-reactivity assessment:

  • Pre-adsorption of antibodies with potential cross-reactive proteins

  • Sequential rather than simultaneous application of antibodies if cross-reactivity is suspected

These controls ensure that observed co-localization patterns reflect genuine biological relationships rather than technical artifacts.

How can researchers quantitatively assess FREM2 expression levels using FITC-conjugated antibodies?

Quantitative assessment of FREM2 expression using FITC-conjugated antibodies can be approached through several methodologies:

For accurate quantification:

• Include calibration standards with known fluorophore concentrations

• Normalize to cell number or tissue area

• Use consistent acquisition parameters across samples

• Account for FITC photobleaching during image acquisition

• Apply background subtraction and threshold consistently

Quantitative western blotting with an anti-FITC antibody (such as the recombinant monoclonal 4L8R5) may also provide a means to assess total FREM2 protein levels in samples where spatial information is not required.

What are the optimized immunohistochemistry protocols for FREM2 antibody, FITC conjugated?

The following optimized protocol is recommended for immunohistochemistry using FREM2 antibody, FITC conjugated:

For FFPE tissue sections:

• Deparaffinization and rehydration:

  • Xylene: 3 changes, 5 minutes each

  • 100% ethanol: 2 changes, 3 minutes each

  • 95%, 70%, 50% ethanol: 3 minutes each

  • Distilled water: 5 minutes

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • Maintain at 95-98°C for 20 minutes

  • Cool to room temperature, 20 minutes

• Permeabilization:

  • 0.1% Triton X-100 in PBS, 10 minutes

  • Wash 3 times with PBS, 5 minutes each

• Blocking:

  • 5% normal serum (matched to host species of other primary antibodies if dual-labeling)

  • 1% BSA in PBS, 1 hour at room temperature

• FREM2 antibody application:

  • Dilute FREM2 antibody, FITC conjugated at 5 μg/ml in blocking buffer

  • Incubate in a humidified chamber overnight at 4°C in the dark

• Washing:

  • PBS-T (0.05% Tween-20 in PBS): 3 washes, 5 minutes each

• Nuclear counterstaining:

  • DAPI (1:1000) for 5 minutes

  • Wash briefly with PBS

• Mounting:

  • Anti-fade mounting medium

  • Seal with nail polish or mounting tape

For optimal results, antibody concentration should be empirically determined for each application and tissue type.

How can researchers troubleshoot weak or absent FITC signal when using FREM2 antibody?

When confronted with weak or absent FITC signal when using FREM2 antibody, researchers should systematically address potential issues:

• Antibody-related factors:

  • Verify antibody integrity by testing with a known positive control

  • Check storage conditions and expiration date

  • Try increasing antibody concentration up to 10 μg/ml

  • Consider using a fresh aliquot to avoid effects of freeze-thaw cycles

• Sample preparation issues:

  • Ensure complete antigen retrieval; extend time or try alternative buffers

  • Verify fixation parameters are compatible with epitope preservation

  • Test different permeabilization conditions if the epitope is intracellular

  • Reduce background autofluorescence by treating sections with sodium borohydride

• Detection system optimization:

  • Protect from light during all steps to prevent FITC photobleaching

  • Use anti-FITC secondary antibodies for signal amplification if necessary

  • Adjust microscope settings (exposure time, gain, laser power) for optimal detection

  • Consider more sensitive detection systems for low-abundance targets

• FREM2 expression considerations:

  • Confirm FREM2 expression in your sample type; it's known to be expressed in skin epithelium and renal epithelia

  • Consider developmental timing, as FREM2 is involved in embryonic development

  • Test positive control tissues with known FREM2 expression

Systematic evaluation of these factors typically resolves signal issues in most experimental contexts.

How can researchers distinguish between specific and non-specific binding of FREM2 antibody, FITC conjugated?

Distinguishing specific from non-specific binding requires multiple validation approaches:

• Pattern consistency analysis:

  • Compare observed staining patterns with published FREM2 localization data

  • Verify consistency with FREM2's known functions in maintaining epithelial integrity and embryonic development

  • Assess subcellular localization (FREM2 should localize to extracellular matrix)

• Experimental validation:

  • Perform peptide competition assays using the immunogen peptide

  • Compare staining patterns with alternative FREM2 antibodies targeting different epitopes

  • Use knockout/knockdown models as negative controls where feasible

  • Implement isotype controls (rabbit IgG-FITC) at matching concentrations

• Tissue-specific considerations:

  • In skin samples, expect FREM2 staining at epithelial-mesenchymal boundaries

  • In kidney samples, FREM2 should be detected in specific nephron segments

  • Verify absence of signal in tissues not expressing FREM2

• Signal characteristics assessment:

  • Specific binding typically shows consistent patterns across biological replicates

  • Non-specific binding often appears as diffuse background or edge artifacts

  • Evaluate signal-to-noise ratio across different antibody dilutions

What are the best practices for quantifying co-localization between FREM2 and other proteins in dual-labeling experiments?

Quantifying co-localization between FREM2 and other proteins requires rigorous methodology:

• Image acquisition optimization:

  • Use confocal microscopy to minimize out-of-focus signal

  • Ensure no cross-talk between fluorescence channels

  • Acquire images at Nyquist sampling rate for optimal resolution

  • Standardize all acquisition parameters across samples

• Quantification metrics:

  • Pearson's correlation coefficient (PCC): measures linear correlation between intensities

  • Manders' overlap coefficient (MOC): quantifies fraction of overlapping signals

  • Object-based methods: count co-localized structures/puncta

  • Intensity correlation analysis (ICA): assesses dependency of intensity variations

• Analysis workflow:

  • Apply appropriate background subtraction

  • Set consistent thresholds using objective methods

  • Use specialized co-localization software (e.g., JACoP plugin for ImageJ)

  • Analyze multiple regions of interest across multiple samples

• Statistical validation:

  • Compare observed co-localization to randomized controls

  • Implement Costes randomization test for statistical significance

  • Report confidence intervals for co-localization metrics

  • Use appropriate statistical tests when comparing experimental groups

For FREM2 studies, co-localization with other extracellular matrix proteins or cell adhesion molecules would be particularly relevant given its functional roles .

How does FREM2 antibody, FITC conjugated, complement other research tools in developmental biology studies?

FREM2 antibody with FITC conjugation provides complementary insights when integrated with other research tools in developmental biology:

• Integration with genetic approaches:

  • Visualization of protein expression in conditional knockout models (e.g., Frem2 fl/fl mice)

  • Correlation of FREM2 protein localization with phenotypes in mutant models

  • Assessment of rescue experiments by monitoring protein re-expression

• Compatibility with tissue clearing techniques:

  • Enables 3D visualization of FREM2 distribution in intact embryonic structures

  • Can be combined with light-sheet microscopy for whole-organ imaging

  • Allows for tracking developmental dynamics of FREM2 expression through transparent tissues

• Multi-omics integration:

  • Correlation of protein localization data with transcriptomic profiles

  • Validation of proteomics findings at the tissue level

  • Integration with interactome data to visualize protein-protein interactions in situ

• Developmental timing studies:

  • Tracking FREM2 expression throughout embryonic development

  • Correlation with developmental milestones in epithelial formation

  • Investigation of FREM2's role in eyelid and anterior eye segment development

FREM2 has been implicated as an essential gene during embryonic development , making these integrated approaches particularly valuable for understanding its developmental functions.

What recent advances have been made in understanding FREM2 function using antibody-based approaches?

Recent research using antibody-based approaches has expanded our understanding of FREM2 function:

• Expanded tissue distribution knowledge:

  • Identification of FREM2 expression in previously uncharacterized tissues

  • Higher resolution mapping of FREM2 distribution within known positive tissues

  • Better understanding of subcellular localization patterns

• Functional insights:

  • FREM2 has been identified as a candidate mechanosensory tether in touch sensation studies, though subsequent investigation focused more on TENM4 as the primary mechanotransduction component

  • Further characterization of its role in maintaining epithelial integrity

  • Enhanced understanding of its developmental functions in eyelid and eye anterior segment formation

• Pathological associations:

  • Correlation of FREM2 expression patterns with developmental abnormalities

  • Investigation of potential altered expression in epithelial disorders

  • Possible involvement in renal pathologies given its role in renal epithelia maintenance

• Technical advancements:

  • Development of more specific antibodies against different FREM2 domains

  • Implementation of antibody-based proximity labeling for identifying interaction partners

  • Improved detection sensitivity through signal amplification methods

Future research directions may include further investigation of FREM2's potential role in mechanosensation and more detailed characterization of its functions in epithelial development and maintenance.