FABP4 Antibody，FITC conjugated

What is FABP4 and why is it an important research target?

FABP4 (Fatty Acid-Binding Protein 4) is a lipid transport protein primarily expressed in adipocytes that plays critical roles in both metabolic and inflammatory pathways . It functions by binding both long chain fatty acids and retinoic acid, then delivering these molecules to their cognate receptors in the nucleus . FABP4 has emerged as an important research target due to its involvement in several pathological conditions including cardiovascular disease, metabolic disorders, and inflammatory conditions such as allergic airway inflammation . Specifically, FABP4 has been shown to regulate eosinophil recruitment and activation in allergic airway inflammation, making it relevant for asthma research .

What are the key applications for FABP4 Antibody, FITC conjugated?

FABP4 Antibody, FITC conjugated is specifically designed for applications requiring fluorescent detection of FABP4 protein. The primary applications include:

• Immunofluorescence (IF): For visualization of FABP4 in tissue sections or cultured cells

• Immunocytochemistry (ICC): For detecting FABP4 in fixed cells

• Immunohistochemistry (IHC): For localizing FABP4 in tissue sections

• Flow cytometry (FACS): For analyzing FABP4 expression in cell populations

The FITC conjugation eliminates the need for secondary antibody incubation steps, streamlining experimental workflows and reducing background noise in fluorescence-based applications .

How should FABP4 Antibody, FITC conjugated be stored and handled?

For optimal performance and longevity, FABP4 Antibody, FITC conjugated should be stored at 2-8°C and should not be frozen . The antibody is typically presented in lyophilized form from PBS pH 7.4 with stabilizers such as 20 mg/ml BSA, 0.02% Sodium Azide, and 4% Trehalose . When working with the antibody, minimize exposure to light as FITC is photosensitive and can photobleach, resulting in decreased signal intensity. For long-term storage, keep the original vial tightly sealed and avoid repeated freeze-thaw cycles which can degrade antibody performance .

How can I validate the specificity of FABP4 Antibody, FITC conjugated?

Validating antibody specificity is critical for reliable experimental results. For FABP4 Antibody, FITC conjugated, consider these validation approaches:

• Positive and negative control tissues/cells: Use tissues known to express FABP4 (e.g., adipose tissue, heart tissue) as positive controls and tissues known not to express FABP4 as negative controls .

• Western blot verification: Before immunostaining experiments, verify antibody specificity via Western blot using recombinant FABP4 protein. The expected band size for FABP4 is approximately 14-15 kDa .

• Competitive binding assay: Pre-incubate the antibody with recombinant FABP4 protein before staining to demonstrate signal reduction.

• Cross-species reactivity testing: Test on both human and mouse samples if working with animal models, as some FABP4 antibodies have demonstrated cross-reactivity with mouse and rat FABP4 .

• Knockout/knockdown controls: If available, use FABP4-knockout or knockdown samples to confirm specificity.

What is the optimal working concentration for FABP4 Antibody, FITC conjugated in different applications?

Optimal working concentrations vary by application and should be determined empirically for each experimental system:

Based on published protocols, 10 μg/mL has been successfully used for immunofluorescence analysis of FABP4 in mixed cell cultures . For Western blotting applications with related non-conjugated FABP4 antibodies, concentrations around 5 μg/mL have been effective . Always perform a titration experiment with your specific samples to determine the optimal concentration that maximizes specific signal while minimizing background.

How can I mitigate background fluorescence when using FABP4 Antibody, FITC conjugated?

Background fluorescence can significantly impact the quality of results when using FITC-conjugated antibodies. To minimize background:

• Optimize blocking conditions: Use 3-5% serum from the species unrelated to the primary antibody host (e.g., for rabbit polyclonal antibodies, use goat or horse serum) in PBS or TBS with 0.1-0.3% Triton X-100 for permeabilization .

• Include appropriate controls: Always include a negative control (omitting primary antibody) to assess autofluorescence and non-specific binding of the detection system.

• Reduce autofluorescence: For tissues with high autofluorescence (e.g., tissues containing lipofuscin), pretreat with 0.1% Sudan Black B in 70% ethanol for 20 minutes, followed by thorough washing.

• Optimize fixation: Overfixation can increase background and reduce antigen accessibility. For FABP4 detection, mild fixation with 4% paraformaldehyde for 15-20 minutes is typically sufficient .

• Use appropriate antigen retrieval: For paraffin-embedded tissues, antigen retrieval methods may be necessary to expose epitopes. Heat-induced epitope retrieval in citrate buffer (pH 6.0) has been successfully used for FABP4 detection .

• Titrate antibody concentration: Use the minimum concentration that gives a clear specific signal.

How can FABP4 Antibody, FITC conjugated be used to study eosinophil recruitment in allergic airway inflammation?

FABP4 has been identified as a key regulator of eosinophil recruitment and activation in allergic airway inflammation . To study this process:

• Dual immunofluorescence staining: Combine FABP4 Antibody, FITC conjugated with antibodies against eosinophil markers (e.g., anti-MBP antibody) to co-localize FABP4 expression specifically in eosinophils .

• Ex vivo eosinophil functional assays: Isolate eosinophils from appropriate sources (e.g., peripheral blood, bronchoalveolar lavage) and assess FABP4 expression in response to inflammatory stimuli such as TNF-α, IL-4, and IL-13, which have been shown to induce FABP4 expression .

• In vitro migration assays: Use FABP4 Antibody, FITC conjugated to track changes in FABP4 expression during eosinophil migration in response to eotaxin-1. Compare wild-type and FABP4-deficient eosinophils to determine how FABP4 impacts migration, F-actin polymerization, and calcium flux .

• Murine models of allergic airway inflammation: In cockroach antigen (CRA)-induced allergic airway inflammation models, use the antibody to track FABP4 expression in recruited inflammatory cells, particularly eosinophils . This approach helps correlate FABP4 expression with markers of inflammation such as IL-5, IL-13, TNF-α, and cysteinyl leukotriene C4 levels .

How does FABP4 expression correlate with ERK(1/2) phosphorylation in inflammatory cells?

Research has demonstrated a regulatory relationship between FABP4 expression and ERK(1/2) phosphorylation in the inflammatory response pathway . To investigate this relationship:

• Western blot analysis: After stimulating cells with appropriate inflammatory mediators (e.g., eotaxin-1), perform Western blot analysis using antibodies against phospho-ERK(1/2) and total ERK(1/2) . Compare FABP4-expressing and FABP4-deficient cells to determine how FABP4 affects ERK(1/2) phosphorylation kinetics.

• Quantitative measurement: Use image analysis software (e.g., ImageJ) to measure band intensity, normalizing phospho-ERK(1/2) signals against total ERK(1/2) .

• Time-course experiments: Establish the temporal relationship between FABP4 expression and ERK(1/2) phosphorylation by collecting samples at multiple time points after stimulation.

• Pharmacological inhibition: Use ERK(1/2) pathway inhibitors in combination with FABP4 detection to establish causality in the signaling pathway.

Results from such experiments have shown that FABP4-deficient eosinophils exhibit decreased ERK(1/2) phosphorylation in response to eotaxin-1 compared to wild-type cells, suggesting that FABP4 positively regulates the ERK(1/2) signaling pathway during inflammatory responses .

What methodologies can be used to study FABP4 regulation at the transcriptional level?

Understanding FABP4 transcriptional regulation provides insights into its role in various pathological conditions. Several methodologies can be employed:

• Quantitative RT-PCR (qRT-PCR): Use qRT-PCR to measure FABP4 mRNA levels in response to various stimuli or in different experimental conditions . Systems such as the iQ5 Multicolor Real-Time PCR Detection System with iTaq Universal SYBR Green Supermix Kit have been successfully employed for FABP4 quantification .

• mRNA analysis protocol:

  • Extract total RNA from cells/tissues of interest

  • Synthesize cDNA using reverse transcriptase

  • Perform qRT-PCR with FABP4-specific primers

  • Calculate relative expression using the ΔΔCt method (2^-ΔΔCt), normalizing to β-actin as an internal control

• Promoter analysis: Investigate the FABP4 promoter region for transcription factor binding sites, particularly focusing on PPAR-γ, which has been implicated in FABP4 regulation .

• ChIP assays: Perform chromatin immunoprecipitation to identify transcription factors that bind to the FABP4 promoter in vivo.

Why might I observe inconsistent staining patterns with FABP4 Antibody, FITC conjugated?

Inconsistent staining patterns can result from various factors:

• Sample preparation variations: Inconsistent fixation times or conditions can affect epitope accessibility. Standardize fixation protocols (typically 4% paraformaldehyde for 15-20 minutes) and perform antigen retrieval when necessary .

• Antibody degradation: FITC conjugates are sensitive to light and pH. Minimize light exposure during storage and handling, and ensure proper storage at 2-8°C .

• Cell-specific expression patterns: FABP4 expression can vary significantly between cell types and is induced during inflammatory conditions . Ensure appropriate positive controls (e.g., adipose tissue) and negative controls are included in each experiment.

• Technical variability: Inconsistent blocking or washing steps can lead to variable background. Standardize protocols and use automated staining systems when possible.

• Cross-reactivity: Ensure the antibody's specificity by verifying it doesn't cross-react with other FABP family members that may be expressed in your samples.

How can I optimize co-staining protocols using FABP4 Antibody, FITC conjugated with other markers?

For successful co-staining experiments:

• Sequential staining approach:

  • Apply the non-FITC conjugated primary antibody first, followed by its appropriate secondary antibody

  • Block with excess unconjugated host species IgG from the same species as the FABP4 antibody

  • Apply the FABP4 Antibody, FITC conjugated

  • This minimizes cross-reactivity between antibodies

• Spectral compatibility: Choose fluorophores with minimal spectral overlap with FITC (excitation ~495 nm, emission ~520 nm). Good companions include:

  • Red fluorophores (e.g., Cy3, Alexa Fluor 594)

  • Far-red fluorophores (e.g., Cy5, Alexa Fluor 647)

  • UV-excitable dyes (e.g., DAPI for nuclear counterstaining)

• Optimization examples: Successful co-staining has been demonstrated with FABP4 Antibody (green) and CD31 antibody (red) in cocultures of HDLECs and C2C12 cells, using appropriate secondary antibodies for detection .

• Controls for spectral bleed-through: Include single-stained controls to verify and correct for any spectral overlap during image acquisition and analysis.

What are the critical factors in quantifying FABP4 expression using image analysis after immunofluorescence?

For accurate quantification of FABP4 expression from immunofluorescence images:

• Image acquisition standardization:

  • Maintain consistent exposure settings between samples

  • Use identical gain and offset values

  • Avoid image saturation which prevents accurate quantification

  • Capture multiple representative fields per sample

• Background correction:

  • Subtract background fluorescence using unstained regions

  • Use rolling ball background subtraction algorithms for uneven backgrounds

• Thresholding approaches:

  • Set consistent threshold values across all samples

  • Consider automated thresholding methods (e.g., Otsu's method) to reduce subjectivity

• Quantification metrics:

  • Mean fluorescence intensity (MFI) within regions of interest

  • Percentage of cells expressing FABP4 above threshold

  • Integrated density (product of area and mean intensity)

• Normalization strategies:

  • Normalize to cell number using nuclear counterstains

  • Use internal reference markers for relative expression analysis

• Statistical validation:

  • Analyze sufficient cells/fields for statistical significance

  • Apply appropriate statistical tests based on data distribution

When quantifying FABP4 in tissue sections, methodology similar to that used for eosinophil-specific major basic protein (MBP) can be applied, where positive cells are counted per field across multiple representative areas .

How can FABP4 Antibody, FITC conjugated be used in studies examining the relationship between FABP4 and cardiovascular disease?

FABP4 has emerged as a biomarker and potential mediator in cardiovascular disease processes . For investigating this relationship:

• Tissue-specific expression analysis:

  • Use FABP4 Antibody, FITC conjugated to examine FABP4 expression in cardiac tissues, blood vessels, and inflammatory cells within atherosclerotic plaques

  • Compare expression patterns between healthy and diseased tissues

  • Western blot analysis of heart tissue samples has demonstrated FABP4 expression, which can be further characterized by immunofluorescence

• Co-localization studies:

  • Perform dual immunofluorescence with markers of:

    • Macrophages (e.g., CD68) to study FABP4 in foam cells

    • Endothelial cells (e.g., CD31) to examine FABP4 in vascular inflammation

    • Adipocytes adjacent to vascular structures

• In vitro models:

  • Examine FABP4 expression in cultured cardiomyocytes, vascular smooth muscle cells, or endothelial cells under conditions mimicking cardiovascular stress

  • Assess how FABP4 levels change in response to hypoxia, oxidative stress, or inflammatory cytokines

• Ex vivo tissue analysis:

  • Analyze FABP4 expression in human cardiac biopsy samples or surgically excised atherosclerotic plaques

  • Compare expression patterns with clinical parameters and patient outcomes

What methodology can be used to investigate the role of FABP4 in cancer research using FITC-conjugated antibodies?

Emerging research indicates FABP4 may play important roles in various cancer types:

• Cancer tissue microarray analysis:

  • Use FABP4 Antibody, FITC conjugated to screen multiple cancer types simultaneously

  • Published research has demonstrated FABP4 staining in bladder cancer tissue, localizing to the cytoplasm

  • Quantify expression levels and correlate with clinicopathological features

• Tumor microenvironment studies:

  • Investigate FABP4 expression in tumor-associated adipocytes and immune cells

  • Perform triple staining with cancer markers, immune cell markers, and FABP4

  • Analyze spatial relationships between FABP4-expressing cells and tumor progression

• Functional analysis protocols:

  • Isolate primary cancer cells or use established cell lines

  • Manipulate FABP4 expression (overexpression or knockdown)

  • Monitor changes in proliferation, migration, invasion, and response to therapy

  • Use FABP4 Antibody, FITC conjugated to track expression changes in real-time during live cell imaging

• Patient-derived xenograft (PDX) models:

  • Establish PDX models from patient tumor samples

  • Track FABP4 expression during tumor growth and metastasis

  • Evaluate responses to treatments targeting FABP4 or related pathways

Notably, immunohistochemical studies using FABP4 antibodies have successfully detected FABP4 in bladder cancer tissue sections with specific localization to the cytoplasm, demonstrating the utility of these antibodies in cancer research .

How can FABP4 Antibody, FITC conjugated be utilized in flow cytometry to analyze FABP4 expression in heterogeneous cell populations?

Flow cytometry offers powerful capabilities for analyzing protein expression at the single-cell level:

• Sample preparation protocol:

  • Harvest cells and create single-cell suspensions

  • Fix cells with 2-4% paraformaldehyde (10-15 minutes at room temperature)

  • Permeabilize with 0.1% saponin or 0.1% Triton X-100 in PBS

  • Block with 3-5% serum from the same species as the secondary antibody

  • Stain with FABP4 Antibody, FITC conjugated (typically 1-5 μg/mL)

  • Co-stain with lineage markers as needed

• Multiparameter analysis strategy:

  • Combine FABP4 staining with antibodies against:

    • Cell type-specific markers (e.g., CD45 for leukocytes, CD3 for T cells)

    • Activation markers (e.g., CD69, CD25)

    • Other functional proteins of interest

  • Use appropriate compensation controls to correct for spectral overlap

• Data analysis approach:

  • Gate on viable cells using appropriate viability dyes

  • Identify cell populations of interest using lineage markers

  • Analyze FABP4 expression within each population

  • Compare mean fluorescence intensity or percent positive cells across experimental conditions

• Sorting applications:

  • Sort FABP4-high and FABP4-low populations for downstream functional assays

  • Isolate specific cell types expressing FABP4 for transcriptomic or proteomic analysis