FOXL2 Antibody, FITC conjugated

What is FOXL2 and what cellular functions does it regulate?

FOXL2 is a transcriptional regulator that serves as a critical factor for ovary differentiation and maintenance. It functions by repressing the genetic program for somatic testis determination and prevents trans-differentiation of ovary to testis through transcriptional repression of the Sertoli cell-promoting gene SOX9. Additionally, FOXL2 has apoptotic activity in ovarian cells and suppresses ESR1-mediated transcription of PTGS2/COX2 stimulated by tamoxifen . FOXL2 also regulates CYP19 expression, activates SIRT1 transcription under cellular stress conditions, activates transcription of OSR2, acts as a transcriptional repressor of STAR, and participates in SMAD3-dependent transcription of FST via the intronic SMAD-binding element .

What are the key applications for FOXL2 antibody, FITC conjugated?

FOXL2 antibody, FITC conjugated is primarily used in several key applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): Typically at dilutions of 1:100-1:500 for detection and quantification of FOXL2 in samples .

• Immunofluorescence: Particularly useful for visualizing FOXL2 expression in tissue sections, especially in ovarian tissue samples .

• Flow Cytometry: Some FOXL2 antibodies can be used for flow cytometric analysis of cells expressing this protein .

The FITC conjugation enables direct visualization under fluorescence microscopy without the need for secondary antibodies, making it particularly valuable for co-localization studies and reducing background in immunofluorescence applications.

What tissues and species show high FOXL2 reactivity with these antibodies?

FOXL2 antibodies show reactivity primarily in:

FOXL2 expression is particularly high in granulosa cells of the ovary, making these tissues ideal for positive controls. The protein is primarily localized in the nucleus of cells due to its function as a transcription factor .

How should I optimize fixation for immunofluorescence with FITC-conjugated FOXL2 antibody?

For optimal immunofluorescence results with FITC-conjugated FOXL2 antibody:

• Fixation: Use 4% paraformaldehyde in PBS for 30 minutes for tissue samples .

• Post-fixation processing: Follow with sequential 30-minute incubations in ice-cold ethanol (50%, followed by 75%) before paraffin embedding .

• Antigen retrieval: For paraffin sections, perform epitope unmasking by boiling in 10mM citric acid, pH 6.0 .

• Blocking: Inactivate endogenous peroxidases using 1.5% hydrogen peroxide, then use appropriate blocking solution (such as those from tyramide signal amplification kits) .

• Dilution range: Start with the recommended range (1:100-1:500) and optimize based on your specific tissue type and fixation protocol .

This methodology has been validated in research examining FOXL2 expression patterns in ovarian development and function.

How can I ensure specificity when using FOXL2 antibody in co-localization studies?

When using FITC-conjugated FOXL2 antibody for co-localization studies:

• Sequential antibody application: For co-localization with other rabbit antibodies (such as TSHβ), use Fab fragment goat anti-rabbit antibody (1:100) for blocking for 30 minutes prior to applying the second primary antibody .

• Controls for spectral overlap: Include single-stained controls to account for potential bleed-through between FITC and other fluorophores.

• Validation with knockout samples: When available, use samples from conditional knockout models (like gonadotrope-specific Foxl2 cKO mice) as negative controls to confirm specificity .

• Cross-reactivity assessment: Test for potential cross-reactivity with FOXL1 or other forkhead box family members that share structural similarities.

• Pre-absorption controls: Consider running parallel staining with antibody pre-absorbed with recombinant FOXL2 protein (specifically the immunogen region 116-216AA) to demonstrate binding specificity .

These approaches help minimize false-positive results and ensure reliable co-localization data, particularly important when studying FOXL2's interaction with the SMAD proteins in activin-mediated signaling pathways.

What are the methodological considerations when using FOXL2 antibody to investigate fertility disorders?

When investigating fertility disorders using FOXL2 antibody:

• Sample selection considerations:

  • Include age-matched controls, as FOXL2 expression changes during development

  • Consider using paired ovarian and pituitary samples, as FOXL2 functions in both tissues

• Fertility phenotype assessment protocol:

  • For female samples: Analyze ovarian weight, follicle counts, ovulation numbers, and FSH levels

  • For male samples: Assess testis size, spermatogenesis parameters, and FSH levels

• Hormonal correlation methodology:

  • Measure serum FSH levels and correlate with FOXL2 staining intensity

  • Examine Fshb mRNA levels in pituitary alongside FOXL2 expression

• Comparative analysis approach:

  • Use natural estrous cycle samples alongside gonadotropin-stimulated samples

  • Compare juvenile vs. adult tissue responses

FOXL2 dysfunction has been linked to blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) with premature ovarian failure, making this antibody valuable for investigating the molecular basis of certain infertility cases .

How does phosphorylation status affect FOXL2 detection and function in experimental models?

FOXL2 phosphorylation significantly impacts both detection and functional analysis:

• Phospho-specific detection: Consider using phospho-specific antibodies (such as those targeting phospho-Ser263) alongside total FOXL2 antibodies to correlate phosphorylation status with activity .

• Sample preparation protocol for preserving phosphorylation:

  • Use phosphatase inhibitors (e.g., sodium orthovanadate, sodium fluoride) in lysis buffers

  • Maintain samples at 4°C throughout processing

  • Consider using phosphatase treatments on parallel samples as controls

• Functional correlation methodology:

  • Phosphorylation affects FOXL2's interaction with SMAD proteins in activin-mediated pathways

  • Compare wild-type and phospho-mutant FOXL2 effects on Fshb transcription

• Stress response consideration:

  • Cellular stress induces changes in FOXL2 phosphorylation affecting SIRT1 transcription

  • Include oxidative stress conditions in experimental designs when studying phosphorylation effects

These considerations are particularly important when studying FOXL2's role in activin-stimulated formation of SMAD protein complexes that drive Fshb transcription in gonadotrope cells.

What are the optimal protocols for investigating FOXL2-mediated repression of SOX9 using FITC-conjugated antibodies?

For studying FOXL2-mediated repression of SOX9:

• Dual immunofluorescence protocol:

  • Deparaffinize and rehydrate tissue sections

  • Perform antigen retrieval in sodium citrate buffer (pH 6.0)

  • Block with 5% normal donkey serum for 1 hour at room temperature

  • Incubate with FITC-conjugated FOXL2 antibody (1:100-1:500) overnight at 4°C

  • Apply SOX9 antibody (raised in a different species than FOXL2 antibody)

  • Use appropriate secondary antibody for SOX9 detection

• Quantitative analysis methodology:

  • Use digital image analysis to quantify nuclear FOXL2 and SOX9 signal intensities

  • Apply colocalization algorithms to assess potential overlapping expression

  • Correlate expression levels with gonadal development stages

• Experimental model selection:

  • Conditional knockout models targeting either FOXL2 or SOX9

  • Time-course studies during gonadal development

  • Ovary-to-testis transdifferentiation models

This approach enables investigation of the critical role FOXL2 plays in maintaining ovarian identity by preventing SOX9 expression, which would otherwise promote testis development .

How can I troubleshoot inconsistent results when using FOXL2 antibody, FITC conjugated in different experimental contexts?

When encountering inconsistent results with FITC-conjugated FOXL2 antibody:

• Fluorophore stability assessment:

  • FITC is sensitive to photobleaching; minimize exposure to light during storage and processing

  • Check fluorescence signal on control samples to verify conjugate integrity

  • Consider storage conditions: maintain antibody at 4°C in the dark for short-term and -20°C for long-term storage

• Systematic optimization protocol:

  • Titration series: Test antibody at multiple dilutions (1:50, 1:100, 1:200, 1:500) on standard samples

  • Fixation comparison: Compare 4% paraformaldehyde with alternative fixatives

  • Antigen retrieval methods: Test multiple pH conditions and heating protocols

• Sample-specific considerations:

  • Age-dependent expression: FOXL2 levels vary during development

  • Tissue-specific optimization: Pituitary versus ovarian tissue may require different protocols

  • Species differences: Human, mouse, and rat samples may require different antigen retrieval approaches

• Technical variability reduction:

  • Standardize image acquisition parameters (exposure time, gain)

  • Include internal control tissues in each experimental run

  • Consider batch effects when comparing data across multiple experiments

These troubleshooting approaches address the most common sources of inconsistency in immunofluorescence experiments with FOXL2 antibodies.

What methodologies are recommended for investigating FOXL2's role in activin-mediated FSH synthesis?

For investigating FOXL2's role in activin-mediated FSH synthesis:

• Experimental design strategy:

  • Compare pituitary FSH levels and Fshb mRNA expression between wild-type and gonadotrope-specific Foxl2 conditional knockout models

  • Assess basal versus activin-stimulated FSH production in both models

• Molecular interaction analysis protocol:

  • Use chromatin immunoprecipitation (ChIP) with FOXL2 antibody to assess binding to the Fshb promoter

  • Investigate FOXL2-SMAD protein complex formation using co-immunoprecipitation followed by Western blotting

  • Examine activin receptor signaling components in relation to FOXL2 expression

• Transcript analysis methodology:

  • Quantify Fshb mRNA using quantitative RT-PCR in response to activin treatment

  • Compare FSH protein levels using specific antibodies against FSH

  • Correlate FOXL2 nuclear localization with FSH production

This approach enables detailed investigation of how FOXL2 participates in SMAD3-dependent transcription pathways, particularly important since FOXL2 deficiency leads to significantly impaired FSH synthesis and consequent fertility issues .

How can I effectively use FOXL2 antibody to study its role in CYP19 regulation and estrogen synthesis?

For studying FOXL2's role in CYP19 regulation and estrogen synthesis:

• Expression correlation analysis:

  • Use dual immunofluorescence to co-localize FOXL2 (using FITC-conjugated antibody) and CYP19 in ovarian granulosa cells

  • Quantify relative expression levels across follicular development stages

  • Correlate expression patterns with estradiol levels

• Promoter interaction study protocol:

  • Perform ChIP assays using FOXL2 antibody to analyze binding to the CYP19 promoter

  • Use reporter gene assays with wild-type and mutant CYP19 promoter constructs

  • Assess the impact of FOXL2 knockdown/overexpression on CYP19 transcription

• Functional consequences methodology:

  • Measure estradiol production in granulosa cells with normal versus altered FOXL2 expression

  • Correlate CYP19 activity with FOXL2 nuclear localization

  • Assess the impact of estrogen synthesis inhibitors on FOXL2 expression as a feedback mechanism

This methodological approach allows for comprehensive analysis of how FOXL2 functions as a regulator of CYP19 expression, which is critical for estrogen synthesis in ovarian follicles .

What are the recommended protocols for investigating FOXL2's role in cellular apoptosis using FITC-conjugated antibodies?

For investigating FOXL2's apoptotic function:

• Apoptosis detection combined with FOXL2 visualization:

  • Perform TUNEL assay or Annexin V staining together with FOXL2 immunofluorescence

  • Use FITC-conjugated FOXL2 antibody (1:100-1:500) followed by a different fluorophore for apoptotic markers

  • Include counterstaining for nuclear visualization (DAPI/Hoechst)

• Quantitative correlation approach:

  • Analyze the percentage of FOXL2-positive cells undergoing apoptosis

  • Compare apoptotic rates in cells with high versus low FOXL2 expression

  • Assess nuclear morphology changes in relation to FOXL2 expression levels

• Mechanistic pathway analysis:

  • Examine pro-apoptotic gene expression (e.g., BAX, BAD) in FOXL2-expressing cells

  • Assess caspase activation in relation to FOXL2 nuclear localization

  • Investigate the effect of apoptotic stimuli on FOXL2 phosphorylation status

• Ovarian context-specific considerations:

  • Compare follicular atresia rates with FOXL2 expression patterns

  • Assess granulosa cell apoptosis in normal versus pathological ovarian samples

  • Correlate with hormonal parameters that influence follicular survival

This approach enables investigation of FOXL2's established apoptotic activity in ovarian cells, which is critical for normal follicular development and atresia .

What are the optimal parameters for flow cytometric analysis using FITC-conjugated FOXL2 antibody?

For flow cytometric analysis with FITC-conjugated FOXL2 antibody:

• Cell preparation protocol:

  • Fix cells in 2-4% paraformaldehyde for 15-20 minutes at room temperature

  • Permeabilize with 0.1-0.5% Triton X-100 or saponin buffer (critical for nuclear antigen access)

  • Block with 5% serum (matching secondary antibody species) for 30 minutes

  • Incubate with FITC-conjugated FOXL2 antibody at 1:100-1:500 dilution

• Instrument setup considerations:

  • Use 488nm laser for FITC excitation

  • Set primary detection channel at 515-545nm

  • Establish compensation parameters if using multiple fluorophores

  • Include single-stained controls for accurate compensation

• Gating strategy:

  • Initial gating on forward/side scatter to identify cell populations

  • Secondary gating on nuclear size/complexity if analyzing mixed cell populations

  • Final analysis based on FITC signal intensity to classify FOXL2-high and FOXL2-low populations

• Controls and validation:

  • Include isotype control antibody (FITC-conjugated)

  • Use positive control (ovarian granulosa cell line) and negative control (testicular cell line)

  • Consider signal-to-noise ratio optimization through titration experiments

This methodology enables quantitative analysis of FOXL2 expression at the single-cell level, particularly valuable for heterogeneous primary tissue samples .

How can I develop a multiplexed immunofluorescence assay incorporating FOXL2 antibody for studying gonadal development?

For developing a multiplexed immunofluorescence assay:

• Panel design strategy:

  • Combine FITC-conjugated FOXL2 antibody with antibodies for:

    • SOX9 (testis determination factor)

    • DMRT1 (male gonadal development)

    • CYP19/aromatase (estrogen synthesis)

    • AMH (ovarian reserve marker)

  • Select fluorophores with minimal spectral overlap (e.g., FITC, Texas Red, Cy5, Cy7)

• Sequential staining protocol:

  • Begin with antigen retrieval in sodium citrate buffer (pH 6.0)

  • Apply Fab fragment blocking between antibodies raised in the same species

  • Use tyramide signal amplification for low-abundance antigens

  • Include nuclear counterstain (DAPI) for cell identification

• Imaging and analysis workflow:

  • Employ multispectral imaging systems for optimal fluorophore separation

  • Use automated image analysis software for quantitative assessment

  • Apply tissue segmentation algorithms to distinguish different cellular compartments

  • Implement colocalization analysis for protein interaction studies

• Validation methodology:

  • Include single-stained controls for each antibody

  • Use fluorescence minus one (FMO) controls to establish gating thresholds

  • Validate with tissues of known expression patterns

This approach enables comprehensive spatial analysis of multiple factors in the gonadal determination pathway alongside FOXL2, providing insight into temporal and spatial relationships during development .

What methodological approaches are recommended for studying FOXL2 post-translational modifications using antibody-based techniques?

For studying FOXL2 post-translational modifications:

• Sequential immunoprecipitation strategy:

  • Primary immunoprecipitation with total FOXL2 antibody

  • Secondary analysis with modification-specific antibodies (phospho-, SUMO-, ubiquitin-specific)

  • Alternative approach: initial enrichment with modification-specific antibodies followed by FOXL2 detection

• Western blot analysis protocol:

  • Use phospho-specific antibodies (e.g., Phospho-Ser263) alongside total FOXL2 antibody

  • Analyze mobility shifts indicative of modifications

  • Employ phosphatase or deubiquitinase treatments on parallel samples as controls

• Cell treatment methodology:

  • Apply activation stimuli (activin, stress inducers) to trigger specific modifications

  • Use inhibitors of specific modification pathways to confirm relationships

  • Analyze temporal dynamics with time-course experiments

• Mass spectrometry validation:

  • Immunoprecipitate FOXL2 from tissues or cell models

  • Perform mass spectrometry to identify and map modification sites

  • Correlate findings with antibody-based detection methods

This comprehensive approach enables detailed characterization of how post-translational modifications regulate FOXL2 function, particularly important for understanding its context-specific activities in ovarian development and function .

How can I optimize FITC-conjugated FOXL2 antibody signal for confocal microscopy in challenging tissue samples?

For optimizing FITC-conjugated FOXL2 antibody signal in challenging samples:

• Sample preparation enhancement:

  • Extend fixation time to 45-60 minutes for fibrous tissues

  • Increase antigen retrieval duration for heavily fixed samples

  • Consider tissue thickness (optimal: 5-7μm sections for adequate antibody penetration)

  • Employ extended blocking (2-3 hours) for high-background tissues

• Signal amplification methods:

  • Implement tyramide signal amplification for low-abundance detection

  • Use biotin-streptavidin systems for additional signal enhancement

  • Consider sequential application of primary antibody to increase binding

  • Extend primary antibody incubation to 48 hours at 4°C for difficult antigens

• Imaging parameter optimization:

  • Increase pixel dwell time to capture more emitted photons

  • Utilize spectral unmixing for samples with high autofluorescence

  • Implement deconvolution algorithms to improve signal-to-noise ratio

  • Apply line averaging (4-8 lines) to reduce random noise

• Quantitative validation approach:

  • Include internal control regions within each sample

  • Establish standard curves with known positive samples

  • Implement batch correction algorithms for multi-session imaging

These methodologies have proven effective for detecting FOXL2 in challenging contexts such as highly fibrotic ovarian tissue or samples with significant autofluorescence .

What are the recommended approaches for quantitative analysis of FOXL2 expression in relation to fertility phenotypes?

For quantitative analysis of FOXL2 expression in fertility studies:

• Standardized image acquisition protocol:

  • Maintain consistent microscope settings across all samples

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

  • Include calibration standards in each imaging session

  • Apply flat-field correction to compensate for illumination variations

• Nuclear expression quantification methodology:

  • Implement nuclear segmentation based on DAPI counterstaining

  • Measure mean fluorescence intensity of FOXL2 within nuclear regions

  • Calculate nuclear/cytoplasmic ratio to assess localization efficiency

  • Classify cells based on expression intensity thresholds

• Correlation with fertility parameters:

  • Analyze relationship between FOXL2 expression levels and:

    • Ovarian follicle counts at different developmental stages

    • FSH and estradiol serum levels

    • Ovulation rates and corpus luteum formation

    • Fshb mRNA levels in pituitary samples

• Statistical analysis approach:

  • Apply hierarchical linear models to account for within-sample correlation

  • Use principal component analysis to identify patterns across multiple parameters

  • Implement regression models to quantify relationships between expression and function

  • Calculate minimum sample sizes based on observed effect sizes and variability

This integrated approach enables robust quantification of how FOXL2 expression patterns correlate with functional fertility outcomes, providing insight into the mechanisms of fertility disorders associated with FOXL2 dysfunction .