FGFR2 Antibody, FITC conjugated

What is FGFR2 and why is it an important research target?

FGFR2 (Fibroblast Growth Factor Receptor 2) is a transmembrane tyrosine kinase that functions as a cell-surface receptor for fibroblast growth factors, playing essential roles in cell proliferation, differentiation, migration, and apoptosis. It is crucial for embryonic development, including normal embryonic patterning, trophoblast function, limb bud development, lung morphogenesis, osteogenesis, and skin development . FGFR2 signaling is particularly important in the regulation of osteoblast differentiation and skeleton development, promoting proliferation in keratinocytes and immature osteoblasts while inducing apoptosis in differentiated osteoblasts . Its dysregulation has been implicated in various developmental disorders and cancers, making it a significant target for fundamental research and therapeutic development.

What are the different isoforms of FGFR2 and how do they affect antibody selection?

FGFR2 exists in multiple isoforms with structural variations that can impact antibody recognition. The extracellular domain includes three Ig-like domains in alpha isoforms or two Ig-like domains in beta isoforms . Additionally, alternative exon usage results in IIIb or IIIc isoforms that differ in their membrane-proximal Ig-like domain . When selecting a FITC-conjugated FGFR2 antibody, researchers should verify which epitopes and isoforms the antibody recognizes. For example, some antibodies are raised against synthetic peptides from specific regions (amino acids 520-570 in human FGFR2 isoform X6 protein) , while others may recognize epitopes common to multiple isoforms. This distinction is critical for experimental design, particularly when studying tissue-specific expression patterns or isoform-specific functions.

What is the optimal protocol for flow cytometry using FGFR2 Antibody, FITC conjugated?

For optimal flow cytometry results with FGFR2 Antibody, FITC conjugated:

• Harvest cells (1×10^6) and wash twice with ice-cold PBS containing 2% FBS

• Resuspend cells in 100 μL of staining buffer (PBS with 2% FBS)

• Add the FITC-conjugated FGFR2 antibody at the appropriate dilution (typically 1:50-1:200 for flow cytometry)

• Incubate for 30 minutes at 4°C in the dark

• Wash cells twice with staining buffer to remove unbound antibody

• Resuspend in 400-500 μL of staining buffer for analysis

• Include appropriate isotype control antibody (FITC-conjugated IgG of the same species) to establish background fluorescence

For KATO-III human gastric carcinoma cell line and similar FGFR2-expressing cells, this protocol has successfully demonstrated specific binding as evidenced by the clear separation between the antibody-stained samples and isotype controls . Protect samples from light throughout the procedure to maintain FITC fluorescence intensity.

How should FGFR2 Antibody, FITC conjugated be stored to maintain optimal activity?

To maintain optimal activity of FGFR2 Antibody, FITC conjugated:

Most FITC-conjugated antibodies maintain activity for approximately 12 months from the date of receipt when stored properly . Unlike some other conjugated antibodies, FITC-conjugated antibodies should not be frozen after reconstitution as this can affect the fluorochrome stability . Aliquoting into smaller volumes upon receipt is recommended to minimize freeze-thaw cycles.

How can dual staining with FGFR2 Antibody, FITC conjugated be optimized to study receptor-ligand interactions?

When designing dual staining experiments to study FGFR2-ligand interactions:

• Antibody panel design: Pair FGFR2 Antibody, FITC conjugated (excitation/emission: 494nm/518nm) with fluorochromes having minimal spectral overlap such as APC (650nm/660nm) or PE (565nm/578nm) for ligand or co-receptor detection

• Sequential staining protocol:

  • First stain with unconjugated anti-FGF ligand antibody

  • Add fluorophore-conjugated secondary antibody

  • Block with excess IgG from the same species as the first primary

  • Add FGFR2 Antibody, FITC conjugated

  • This prevents cross-reactivity between antibodies

• Compensation controls:

  • Single-stained controls for each fluorochrome

  • FMO (Fluorescence Minus One) controls to establish gating boundaries

  • Isotype controls for both FITC and second fluorochrome

• Receptor-ligand interaction assessment:

  • Fixed ratio analysis: Calculate the ratio of FGFR2:ligand across different experimental conditions

  • Proximity ligation assay: Combine with flow cytometry to detect genuine receptor-ligand complexes

  • FRET (Fluorescence Resonance Energy Transfer): If using compatible fluorophore pairs

This approach has been successfully employed to examine the interaction between FGFR2 and its ligands in gastric carcinoma cells, demonstrating activation of downstream signaling cascades .

What strategies can overcome signal interference when using FGFR2 Antibody, FITC conjugated in tissues with high autofluorescence?

When working with tissues exhibiting high autofluorescence (such as liver, kidney, or brain):

• Tissue-specific autofluorescence reduction:

  • Treat sections with 0.1-1% sodium borohydride for 10 minutes

  • Incubate with 0.1-0.3% Sudan Black B in 70% ethanol for 20 minutes

  • Use commercial autofluorescence quenching reagents specific for aldehyde-fixed tissues

• Advanced imaging techniques:

  • Spectral unmixing: Acquire full emission spectra and computationally separate FITC signal from autofluorescence

  • Time-gated imaging: Exploit the longer lifetime of FITC compared to autofluorescence

  • Confocal microscopy with narrow bandpass filters: Restrict detection to FITC's peak emission wavelength

• Alternative detection strategies:

  • Consider using FGFR2 antibodies conjugated to fluorophores in the far-red spectrum (e.g., Alexa Fluor 647)

  • Sequential immunohistochemistry with FITC quenching between rounds

  • Use of CyTOF technology for mass cytometry detection

• Quantification adjustment:

  • Always subtract background autofluorescence values from neighboring unstained tissue

  • Apply tissue-specific correction factors based on known autofluorescence patterns

  • Employ computational image analysis with machine learning algorithms for signal separation

These approaches have significantly improved signal-to-noise ratios in challenging tissues where FGFR2 expression needed to be accurately quantified.

How can phosphorylation-specific activation of FGFR2 be measured in conjunction with total FGFR2 using FITC-conjugated antibodies?

To simultaneously assess FGFR2 activation status and total expression levels:

• Dual antibody approach:

  • Use FGFR2 Antibody, FITC conjugated for total receptor detection

  • Pair with a phospho-specific antibody against phosphorylated FGFR2 (Y653/654) conjugated to a spectrally distinct fluorophore

  • Apply after appropriate stimulation with FGF ligands

• Flow cytometry protocol refinement:

  • Include phosphatase inhibitors (2mM sodium orthovanadate, 10mM sodium fluoride) in all buffers

  • Perform fixation with 4% paraformaldehyde followed by permeabilization with 90% ice-cold methanol

  • Stain for phospho-FGFR2 first, followed by total FGFR2

  • Calculate activation index as the ratio of phospho-FGFR2 to total FGFR2

• Signaling dynamics assessment:

  • Perform time-course experiments following FGF stimulation (0, 5, 15, 30, 60 minutes)

  • Create phosphorylation kinetics curves normalized to total FGFR2 expression

  • Include inhibitor controls (e.g., FGFR-specific tyrosine kinase inhibitors)

• Downstream signaling correlation:

  • Include detection of phosphorylated downstream targets like PLCG1, FRS2, and PAK4

  • Develop a hierarchical signaling profile to understand sequential activation

This methodology provides quantitative assessment of receptor activation status while controlling for variations in total receptor expression, offering deeper insights into signaling dynamics than either measurement alone.

What are the key considerations when using FGFR2 Antibody, FITC conjugated for detecting rare cell populations?

When investigating rare FGFR2-expressing cell populations (frequency <0.1%):

• Sample enrichment strategies:

  • Negative selection to remove abundant cell types

  • Density gradient centrifugation to isolate specific cell fractions

  • Magnetic pre-enrichment of cells expressing markers co-expressed with FGFR2

• Staining optimization:

  • Higher antibody concentrations may be necessary (1:50 dilution)

  • Extended incubation times (45-60 minutes at 4°C)

  • Addition of blocking reagents to reduce non-specific binding

  • Use of biotin-streptavidin amplification systems for enhanced sensitivity

• Flow cytometry refinements:

  • Collect more events (minimum 1×10^6, ideally 5-10×10^6)

  • Lower flow rate for improved resolution

  • Sequential gating strategy using additional markers

  • Inclusion of viability dye to exclude dead cells

  • Implementation of "dump channel" with lineage markers

• Statistical validation:

  • Calculate minimum event count for statistical significance

  • Perform replicate samples to establish variability

  • Include fluorescence-minus-one (FMO) controls for accurate gating

  • Apply rare event statistical algorithms

This approach has been successfully employed to identify and characterize rare FGFR2-positive progenitor cell populations in developmental studies and cancer stem cell research.

How do differences between monoclonal and polyclonal FGFR2 Antibody, FITC conjugated affect experimental outcomes?

The choice between monoclonal and polyclonal FGFR2 antibodies significantly impacts experimental results:

For quantitative analysis requiring high reproducibility and specificity, monoclonal antibodies like clone 98739 are preferred. For detection of FGFR2 in tissues with potentially masked epitopes or when studying multiple species, polyclonal antibodies may provide advantages. When reporting results, researchers should clearly specify which type was used as this fundamentally affects data interpretation and cross-study comparisons.

What controls should be included when validating FGFR2 Antibody, FITC conjugated for a new application?

A comprehensive validation protocol for FGFR2 Antibody, FITC conjugated should include:

• Positive and negative cell/tissue controls:

  • Positive: KATO-III human gastric carcinoma cell line (known FGFR2 expression)

  • Negative: Cell lines with confirmed absence of FGFR2 expression

  • Gradient controls: Cell lines with varying FGFR2 expression levels

• Technical controls:

  • Isotype control: FITC-conjugated IgG from same species (rabbit or mouse)

  • Fluorescence-minus-one (FMO) controls

  • Secondary antibody-only control (for indirect methods)

  • Unstained control for autofluorescence assessment

• Specificity verification:

  • Blocking peptide competition assay using the immunizing peptide

  • siRNA or CRISPR knockout validation in relevant cell lines

  • Western blot correlation with flow cytometry results

  • Comparison with other validated anti-FGFR2 antibodies

• Dilution optimization:

  • Titration series (1:10, 1:50, 1:100, 1:200, 1:500, 1:1000)

  • Signal-to-noise ratio calculation for each dilution

  • Determination of optimal concentration (typically 1:50-1:200 for flow cytometry)

• Performance metrics documentation:

  • Coefficient of variation across replicates

  • Limit of detection calculation

  • Dynamic range assessment

  • Staining index calculation

This validation workflow ensures reliable, reproducible results and should be thoroughly documented in any publication's methods section.

How can inconsistent staining patterns with FGFR2 Antibody, FITC conjugated be resolved and interpreted?

When encountering inconsistent staining patterns with FGFR2 Antibody, FITC conjugated:

• Common causes and solutions:

  Issue	Potential Cause	Resolution Strategy
  Weak or no signal	Antibody degradation	Check storage conditions; use fresh aliquot
  	Low FGFR2 expression	Increase antibody concentration; try signal amplification
  	Epitope masking	Optimize antigen retrieval; try alternative fixation
  High background	Non-specific binding	Increase blocking; reduce antibody concentration
  	Autofluorescence	Use appropriate quenching reagents; spectral unmixing
  	FITC instability	Protect from light; prepare fresh dilutions
  Variable cell-to-cell staining	Heterogeneous expression	Single-cell analysis; confirm with alternative methods
  	Cell cycle dependence	Synchronize cells; co-stain with cell cycle markers

• Biological interpretation considerations:

  • FGFR2 expression is developmentally regulated and tissue-specific

  • Alternative splicing creates isoforms with different antibody recognition

  • FGFR2 can be internalized upon ligand binding, affecting membrane staining

  • Receptor shedding may occur in certain conditions

• Methodological approaches:

  • Sequential staining with independent FGFR2 antibodies recognizing different epitopes

  • Correlation with mRNA expression by combining with RNA-FISH

  • Time-course experiments to detect dynamic changes in receptor localization

  • Use multiple detection methods (e.g., flow cytometry and immunofluorescence)

These systematic troubleshooting approaches help distinguish between technical artifacts and true biological variability in FGFR2 expression patterns.

How can FGFR2 Antibody, FITC conjugated be integrated into CyTOF (mass cytometry) workflows?

While traditional FITC-conjugated antibodies are designed for fluorescence detection, researchers can leverage FGFR2 antibodies in CyTOF workflows through these approaches:

• Indirect metal labeling:

  • Primary staining with unconjugated FGFR2 antibody (same clone as FITC-conjugated version)

  • Secondary staining with metal-conjugated anti-species antibody

  • This maintains epitope recognition while adapting to CyTOF platform

• Direct antibody metal conjugation:

  • Custom metal conjugation of the same FGFR2 antibody clone (e.g., clone 98739)

  • Use of commercial conjugation kits for metal attachment

  • Validation against standard FITC results to ensure comparable binding properties

• Optimized CyTOF panel design:

  • Include FGFR2 in panels with signaling molecules like phosphorylated PLCG1, FRS2, and MAPK pathway components

  • Combine with markers for cell differentiation status

  • Include proliferation markers to correlate with FGFR2's role in cell proliferation

• Data analysis considerations:

  • Apply dimensionality reduction techniques (tSNE, UMAP)

  • Implement clustering algorithms to identify cell populations

  • Perform trajectory analysis to map developmental pathways

This integration enables simultaneous measurement of FGFR2 expression alongside dozens of other parameters without fluorescence spectral overlap limitations, providing unprecedented insight into FGFR2's role in complex cellular systems.

What are the methodological differences when using FGFR2 Antibody, FITC conjugated for in vivo imaging compared to in vitro applications?

Adapting FGFR2 Antibody, FITC conjugated from in vitro to in vivo imaging requires several methodological adjustments:

• Biological considerations:

  • Blood half-life: FITC-conjugated antibodies typically have 4-8 hour circulation time

  • Tissue penetration: Limited by antibody size (150 kDa) and FITC's excitation/emission properties

  • Target accessibility: Only extracellular epitopes of FGFR2 are accessible in vivo

  • Background autofluorescence: Significantly higher in vivo, particularly in certain tissues

• Technical adaptations:

  • Dosage optimization: Typically 1-5 μg/g body weight (much higher than in vitro concentrations)

  • Administration route: Intravenous injection for systemic imaging; local injection for specific tissues

  • Timing: Peak signal-to-background ratio typically occurs 24-48 hours post-injection

  • Imaging windows: May require surgical preparation for deep tissue visualization

• Alternative approaches:

  • Consider near-infrared fluorophore conjugates instead of FITC for better tissue penetration

  • Use F(ab')2 or Fab fragments for improved pharmacokinetics

  • Employ two-photon microscopy for deeper tissue penetration

  • Combine with tissue clearing techniques for ex vivo imaging

• Controls and validation:

  • Include blocking studies with unlabeled antibody

  • Perform ex vivo tissue analysis to confirm in vivo observations

  • Use FGFR2-knockout or knockdown models as negative controls

These considerations are essential when transitioning from controlled in vitro environments to the complexities of in vivo systems, particularly when studying FGFR2's role in developmental processes or tumor growth.

How can FGFR2 Antibody, FITC conjugated be used to study receptor internalization and trafficking dynamics?

To investigate FGFR2 internalization and trafficking using FITC-conjugated antibodies:

• Pulse-chase experimental design:

  • Pulse: Incubate live cells with FGFR2 Antibody, FITC conjugated at 4°C (prevents internalization)

  • Wash: Remove unbound antibody

  • Chase: Warm cells to 37°C with or without FGF ligands

  • Fix at various timepoints (0, 5, 15, 30, 60, 120 minutes)

• Subcellular colocalization analysis:

  • Co-stain with markers for different endocytic compartments:

    • Early endosomes: Anti-EEA1

    • Recycling endosomes: Anti-Rab11

    • Late endosomes: Anti-Rab7

    • Lysosomes: Anti-LAMP1

  • Quantify colocalization using Pearson's or Mander's coefficients

• Live-cell imaging optimization:

  • Use spinning disk confocal microscopy for rapid acquisition

  • Implement temperature control systems

  • Add minimal essential medium with reduced phenol red

  • Employ computational tracking algorithms for vesicle movement

• Quantitative assessment metrics:

  • Internalization rate: Measure decrease in surface FITC signal over time

  • Recycling rate: Measure reappearance of FITC signal at membrane

  • Degradation kinetics: Measure total FITC signal decrease

  • Calculate half-lives for each process

This approach provides quantitative insights into how FGFR2 trafficking is regulated in normal development and how it may be dysregulated in pathological conditions, with implications for understanding receptor downregulation mechanisms and potential therapeutic interventions.

What methodological approaches can resolve contradictory FGFR2 expression data between antibody-based detection and mRNA analysis?

When faced with discrepancies between FGFR2 protein detection using FITC-conjugated antibodies and mRNA expression data:

• Integrated validation approach:

  • Simultaneous protein and mRNA detection:

    • Flow-FISH technology combining antibody staining with RNA-FISH

    • Sequential immunofluorescence and in situ hybridization

    • Single-cell proteogenomic analysis

• Technical reconciliation strategies:

  • Antibody validation with multiple clones recognizing different FGFR2 epitopes

  • Confirmation with non-antibody methods (e.g., receptor-ligand binding assays)

  • Western blot correlation with different antibody clones

  • Genetic validation using CRISPR-Cas9 knockout controls

• Biological explanations for discrepancies:

  • Post-transcriptional regulation (miRNAs, RNA-binding proteins)

  • Protein stability and half-life differences

  • Alternative splicing affecting antibody epitope recognition

  • Spatial segregation of mRNA versus protein within cells

  • Temporal delays between transcription and translation

• Statistical analysis framework:

  • Correlation analysis between protein and mRNA at single-cell level

  • Bayesian integration of multiple measurement modalities

  • Machine learning approaches to identify patterns in discrepant results

  • Meta-analysis of published literature for similar discrepancies

This methodological framework helps researchers distinguish between technical artifacts and genuine biological phenomena, leading to more accurate interpretation of FGFR2 expression patterns in development and disease.

What emerging technologies might replace or complement FITC conjugation for FGFR2 detection in future research?

The landscape of FGFR2 detection is evolving with several emerging technologies that may supersede or complement traditional FITC conjugation:

• Advanced fluorophore alternatives:

  • Quantum dots: Higher photostability, narrower emission spectra

  • Silicon rhodamines: Far-red emission with minimal autofluorescence overlap

  • Polymer-based fluorophores: Enhanced brightness and stability

  • Lanthanide chelates: Time-resolved fluorescence capabilities

• Proximity-based detection methods:

  • Proximity Extension Assay (PEA): Oligonucleotide-antibody conjugates

  • Fluorescence Resonance Energy Transfer (FRET) pairs

  • NanoBiT complementation for protein-protein interaction studies

  • Luminescent Oxygen Channeling Immunoassay (LOCI)

• Label-free approaches:

  • Surface Plasmon Resonance imaging for receptor dynamics

  • Mass spectrometry imaging for in situ protein detection

  • Raman microspectroscopy for chemical fingerprinting

  • Interferometric detection methods

• Next-generation reporters:

  • CRISPR-based endogenous tagging of FGFR2

  • Split fluorescent protein complementation

  • Photoactivatable fluorescent proteins for super-resolution imaging

  • Genetically encoded biosensors for FGFR2 activation

These technologies promise higher sensitivity, multiplexing capabilities, and dynamic range compared to traditional FITC conjugation, potentially revolutionizing our understanding of FGFR2 biology in development, homeostasis, and disease.

How can conflicting results from different conjugated FGFR2 antibodies (FITC vs. APC vs. Alexa Fluor 647) be systematically resolved?

When confronted with discrepancies between different fluorophore-conjugated FGFR2 antibodies:

• Systematic cross-comparison protocol:

  • Side-by-side staining with identical conditions except for the conjugated antibody

  • Fluorophore normalization using quantitative beads

  • Matched concentration titration experiments

  • Cross-blocking experiments to determine epitope overlap

• Technical factor analysis:

  • Fluorophore physicochemical properties:

    • FITC: pH sensitive, moderate photostability

    • APC: Larger protein, potential steric hindrance

    • Alexa Fluor 647: Higher photostability, less pH sensitive

  • Conjugation chemistry differences affecting antibody avidity

  • Fluorophore-to-protein ratio variations

  • Different clones with distinct epitope recognition

• Multiparametric validation strategy:

  • RNA-scope or qPCR correlation to establish "ground truth"

  • Unconjugated primary antibody with different secondary detection systems

  • Functional assays (phosphorylation, internalization) with each conjugate

  • Cross-validation with orthogonal techniques (Western blot, ELISA)

• Standardized reporting framework:

  • Document conjugation method and fluorophore-to-protein ratio

  • Report antibody concentration in molar terms rather than dilution

  • Specify exact clone, epitope, and binding characteristics

  • Include all tested conditions in supplementary materials