FNDC5 Antibody, FITC conjugated

What is FNDC5 and how does it relate to irisin in experimental contexts?

FNDC5 is a membrane protein that is cleaved and secreted as the hormone irisin. While mouse studies initially suggested FNDC5 expression was primarily exercise-induced in skeletal muscle, recent research has revealed that in brown adipose tissue (BAT), FNDC5 expression is promoted by cold exposure rather than exercise . Understanding this distinction is crucial when designing experiments targeting different tissue types. Methodologically, researchers should consider tissue-specific induction factors when planning experiments to maximize FNDC5 detection - using exercise protocols for muscle tissue studies and cold exposure paradigms (typically 4°C for 4-6 hours) for BAT investigations.

What applications are most suitable for FNDC5 antibodies conjugated to FITC?

FNDC5 antibodies conjugated to FITC are primarily optimized for fluorescence-based applications including:

• Flow cytometry for cellular expression analysis

• Fluorescence microscopy for tissue localization

• Immunohistochemistry of frozen tissue sections

• Fluorescence-activated cell sorting (FACS)

When designing experiments, researchers should note that FITC-conjugated antibodies (such as ABIN7152897) targeting specific amino acid sequences (like AA 113-127) allow for direct visualization without secondary antibodies, simplifying protocols and reducing background . For optimal results in microscopy applications, use appropriate mounting media containing anti-fade reagents to prevent photobleaching.

What are the critical storage and handling considerations for maintaining FNDC5-FITC antibody activity?

FNDC5 antibodies conjugated to FITC require specific storage conditions to maintain fluorophore and epitope integrity. Store at -20°C or -80°C and avoid repeated freeze-thaw cycles . Aliquoting upon receipt is recommended, though some formulations with 50% glycerol may not require aliquoting for -20°C storage . When handling, minimize exposure to light to prevent photobleaching of the FITC fluorophore, and use amber tubes for storage. Reconstitution should follow manufacturer recommendations - typically in PBS containing preservatives like 0.03% ProClin or 0.02% sodium azide . Working solutions should be prepared fresh and kept on ice, protected from light during experimental procedures.

How do FNDC5 antibodies perform across different species in comparative studies?

FNDC5 antibodies show variable cross-reactivity depending on the specific clone and epitope targeted. Many commercially available antibodies demonstrate reactivity with human, mouse, and rat samples . This cross-reactivity facilitates comparative studies across species, which is particularly valuable when investigating evolutionary conservation of FNDC5/irisin function. When designing multi-species experiments, researchers should verify the specific amino acid sequence conservation at the epitope region. For instance, antibodies targeting the AA 113-127 region may have specific human reactivity , while others may offer broader species coverage . Preliminary validation using positive control tissues from each species is essential before proceeding with full experimental designs.

What methodological approaches should be employed to investigate FNDC5's dual localization patterns?

FNDC5 exists both as a membrane-bound precursor and as cleaved, secreted irisin, presenting unique challenges for comprehensive detection. To distinguish between these forms:

• For membrane-bound FNDC5: Use membrane fractionation protocols followed by western blotting, or perform flow cytometry on non-permeabilized cells using FITC-conjugated antibodies that target extracellular domains.

• For secreted irisin: Implement protein precipitation techniques from culture media or biological fluids followed by western blotting or ELISA.

• For co-localization studies: Employ double immunofluorescence with FITC-conjugated FNDC5 antibodies alongside markers for specific cellular compartments (plasma membrane, endoplasmic reticulum, Golgi).

In flow cytometry applications, FNDC5-FITC antibodies have successfully detected the protein in HepG2 human cell lines , with optimal results achieved using standardized protocols for membrane protein staining. When performing immunohistochemistry, clear membrane and cytoplasmic staining patterns have been observed in muscle cells using appropriate concentrations (typically 25 μg/mL) with overnight incubation at 4°C .

How can researchers troubleshoot inconsistent FNDC5 detection results between different tissue types?

Inconsistent detection of FNDC5 across tissues often stems from tissue-specific expression patterns and extraction challenges. To optimize results:

• Tissue-specific optimization: Different tissue types require tailored extraction and staining protocols. For BAT, which shows high FNDC5 expression after cold exposure, use specific extraction buffers containing protease inhibitors designed for adipose tissue .

• Antigen retrieval methods: For IHC applications, compare both citrate buffer (pH 6.0) and TE buffer (pH 9.0) for optimal epitope exposure, as different tissues respond differently to these methods .

• Background reduction strategies: Implement tissue-specific blocking procedures. For adipose tissues, extended blocking (2+ hours) with BSA/serum combinations may be necessary to reduce non-specific binding.

• Sequential dilution series: When transitioning between tissue types, perform antibody dilution series (e.g., 1:200, 1:400, 1:800 for IHC applications) to identify optimal concentrations for each tissue .

• Signal amplification considerations: For tissues with lower expression levels, consider using tyramide signal amplification systems compatible with FITC fluorescence to enhance detection sensitivity.

What experimental design principles should guide investigations of FNDC5/irisin-mediated signaling pathways?

When investigating FNDC5/irisin signaling cascades, particularly the FAK-dependent pathway that activates RUNX1/2 transcription factors:

• Temporal analysis: Design time-course experiments (typically 5, 15, 30, 60 minutes) to capture the kinetics of FAK phosphorylation following exposure to purified irisin, as FAK activation occurs rapidly through autophosphorylation mechanisms .

• Pathway inhibition controls: Include FAK inhibitors (such as PF562271) in parallel experiments to confirm pathway specificity, as this approach has been demonstrated to impair irisin's effects on osteoblast differentiation and RUNX2 transactivation .

• Downstream verification: Implement promoter-reporter assays (such as luciferase assays with the OCN gene promoter) to quantify transcriptional outcomes of pathway activation .

• Protein complex analysis: For investigating protein interactions in the RUNX1/2-PRDM16 complex formation, design co-immunoprecipitation protocols optimized for nuclear proteins, followed by western blotting with specific antibodies against complex components .

• Genetic manipulation approaches: Include experiments with constitutively active FAK mutants (K38A) as positive controls when studying pathway activation, as these have been shown to stimulate RUNX2 transcriptional activity .

What are the optimal protocols for detecting FNDC5 in brown adipose tissue versus skeletal muscle?

Brown adipose tissue (BAT) and skeletal muscle display distinct FNDC5 expression patterns requiring tailored experimental approaches:

For BAT:

• Induction protocol: Cold exposure (4°C for 4-6 hours) maximizes FNDC5 expression in BAT through PGC1α and thyroid hormone receptor cooperation on the FNDC5 gene promoter .

• Extraction buffer: Use specialized adipose tissue extraction buffers containing higher detergent concentrations (1-2% Triton X-100) to effectively solubilize membrane proteins.

• Fixation protocol: For immunofluorescence, brief fixation (10-15 minutes) with 4% paraformaldehyde preserves antigenicity while maintaining tissue integrity.

• Background control: Implement additional blocking steps with normal serum (5-10%) to reduce the high background often encountered in adipose tissues.

For Skeletal Muscle:

• Cross-section preparation: Prepare 8-10 μm cryosections to allow optimal antibody penetration while maintaining tissue architecture.

• Signal amplification: Standard protocols without additional amplification are typically sufficient, as skeletal muscle shows robust FNDC5 expression.

• Counterstaining: Use DAPI nuclear counterstain to clearly visualize the relationship between FNDC5 staining and muscle fiber organization .

How can researchers effectively quantify changes in FNDC5/irisin expression following experimental interventions?

For rigorous quantification of FNDC5/irisin expression changes:

• Multi-method validation: Implement complementary approaches (western blotting, qPCR, ELISA, and immunofluorescence) to confirm expression changes across different methodological platforms.

• Reference gene selection: For qPCR analysis of FNDC5 mRNA, validate multiple reference genes specific to the tissue under investigation, as common housekeeping genes may vary under experimental conditions like exercise or cold exposure.

• Densitometric analysis: For western blot quantification, use standardized loading controls appropriate for the tissue type. For muscle tissue, consider α-actin; for BAT, use UCP1 as an internal control for brown adipocyte content.

• Immunofluorescence quantification: Employ automated image analysis software with standardized parameters for:

  • Mean fluorescence intensity

  • Membrane/cytoplasm signal ratio

  • Percent positive cells

• Statistical approaches: Apply appropriate statistical tests based on data distribution, with paired analyses for before/after interventions and ANOVA for multi-group comparisons with post-hoc corrections for multiple comparisons.

What experimental designs best elucidate FNDC5's role in muscle-adipose-bone connectivity?

To investigate FNDC5's role in inter-organ communication:

• Co-culture systems: Implement transwell co-culture systems with:

  • Muscle cells in the upper chamber

  • Adipocytes or osteoblasts in the lower chamber

  • FNDC5-neutralizing antibodies as intervention controls

• Conditioned media approaches: Collect conditioned media from exercise-stimulated myocytes or cold-exposed brown adipocytes for treatment of osteoblast cultures, with FNDC5 immunodepletion as a control condition.

• Tissue-specific knockout models: Design experiments with tissue-specific FNDC5 knockout models to assess the specific contribution of muscle-derived versus BAT-derived FNDC5/irisin.

• Transcriptional profiling: Implement RNA-seq analysis of target tissues following FNDC5/irisin treatment, focusing on genes associated with:

  • RUNX1/2 transcriptional targets in osteoblasts

  • UCP1 and thermogenesis-related genes in subcutaneous white adipose tissue

  • WW domain-containing protein 2 (WWP2) dependent pathways

• Metabolic phenotyping: Combine metabolic cage analysis with tissue-specific FNDC5 manipulations to correlate inter-organ effects with systemic metabolic parameters.

How should researchers approach the investigation of FNDC5's dual regulation by PGC1α and thyroid hormone receptors?

For studying the transcriptional regulation of FNDC5:

• Promoter analysis: Design luciferase reporter assays incorporating the FNDC5 promoter region containing thyroid hormone response elements (TREs), with mutation analysis of specific binding sites .

• Chromatin immunoprecipitation (ChIP): Implement ChIP protocols optimized for detecting PGC1α and thyroid hormone receptor binding to the FNDC5 promoter in different physiological states (basal, cold-exposed, exercise-stimulated).

• Signaling pathway dissection: Include experiments with PKA pathway inhibitors (such as H89) to assess the contribution of β3-adrenergic receptor signaling to FNDC5 transcription in BAT, as PKA inhibition has been shown to significantly reduce FNDC5 mRNA levels in cold-activated BAT .

• Protein complex analysis: Design co-immunoprecipitation experiments to characterize the composition of the transcriptional complex forming on the FNDC5 promoter, focusing on the interaction between PGC1α and thyroid hormone receptors.

• Hormone manipulation studies: Include T3/T4 supplementation or thyroid hormone receptor antagonist treatments in experimental designs to directly assess the contribution of thyroid hormone signaling to FNDC5 expression in different tissues.

What controls are essential when using FNDC5-FITC antibodies in flow cytometry and immunofluorescence applications?

For rigorous experimental design with FNDC5-FITC antibodies:

• Antibody controls:

  • Isotype control matching the host species and immunoglobulin class (e.g., Rabbit IgG-FITC)

  • Concentration-matched to the primary antibody

  • Processed identically to experimental samples

• Cellular controls:

  • Positive control tissues/cells (skeletal muscle or HepG2 cells known to express FNDC5)

  • Negative control tissues (tissues with minimal FNDC5 expression)

  • Competitive blocking with immunizing peptide where available

• Technical controls for flow cytometry:

  • Single-stained controls for compensation when using multiple fluorophores

  • Unstained cells to establish autofluorescence baseline

  • Fixation-only controls to assess fixation-induced fluorescence

• Signal validation approaches:

  • Antibody titration series to determine optimal concentration

  • Comparison with unconjugated primary + FITC-secondary antibody approach

  • Signal verification using alternative FNDC5 antibodies targeting different epitopes

How can researchers distinguish true FNDC5 signal from background in challenging tissue types?

For optimizing signal-to-noise ratio in FNDC5 detection:

• Autofluorescence reduction strategies:

  • Sudan Black B treatment (0.1-0.3%) for tissues with high autofluorescence (particularly adipose tissue)

  • Photobleaching preprocessing for highly autofluorescent tissues

  • Spectral unmixing during image acquisition when using confocal microscopy

• Background minimization approaches:

  • Extended blocking protocols (2+ hours) with combinations of normal serum, BSA, and non-fat milk

  • Detergent optimization in wash buffers (0.1-0.3% Triton X-100 or Tween-20)

  • Pre-adsorption of antibodies with tissue homogenates from non-target tissues

• Signal amplification considerations:

  • Tyramide signal amplification for tissues with low FNDC5 expression

  • High-sensitivity detection systems (photomultiplier tubes, electron-multiplying CCDs)

  • Extended exposure times with anti-fade reagents to capture weak signals

• Analytical approaches:

  • Background subtraction algorithms during image analysis

  • Ratiometric analysis comparing specific signal to background regions

  • Threshold determination based on isotype control staining intensity