ANGPTL4 Antibody，FITC conjugated

What are the optimal fixation methods for using ANGPTL4 antibodies in immunohistochemistry?

For optimal results with ANGPTL4 antibodies in paraffin-embedded sections, heat-mediated antigen retrieval in EDTA buffer (pH 8.0) is recommended. Paraformaldehyde (PFA) is the preferred fixation method due to its superior tissue penetration capabilities. It's important to note that PFA should be prepared fresh before use, as long-term stored PFA converts into formalin as the molecules aggregate . For immunohistochemical analysis of human tissues, researchers have successfully used protocols involving:

• Tissue section blocking with 10% goat serum

• Incubation with 2 μg/ml anti-ANGPTL4 antibody overnight at 4°C

• Peroxidase-conjugated secondary antibodies incubated for 30 minutes at 37°C

• Development using DAB as the chromogen

This approach has been validated for human placenta and spleen tissues with consistent results.

How can I confirm the specificity of my ANGPTL4 antibody?

Validation of ANGPTL4 antibody specificity requires multiple complementary approaches:

• Blocking peptide experiments: Use the specific immunogenic peptide that was used to generate the antibody to competitively inhibit antibody binding. Many suppliers offer matching blocking peptides for this purpose .

• Multiple detection methods: Validate across different techniques (Western blot, IHC, IF) to ensure consistent detection patterns.

• Positive and negative controls: Include tissues known to express high levels of ANGPTL4 (placenta, liver, adipose tissue) and tissues with negligible expression.

• Cross-reactivity testing: If working across species, validate the antibody in each target species separately, as cross-reactivity cannot be assumed (e.g., antibodies targeting human ANGPTL4 may not necessarily work in primate tissues despite sequence homology) .

What techniques are most effective for studying ANGPTL4 interactions with extracellular matrix proteins?

Research has established that ANGPTL4 interacts with extracellular matrix proteins, particularly vitronectin and fibronectin. For studying these interactions, several advanced techniques have proven effective:

Surface Plasmon Resonance (SPR):

• Immobilize purified cANGPTL4 onto a CM5-carboxylated dextran sensor chip using amine coupling

• Introduce matrix proteins at multiple concentrations (typically 0.16-5.0 μM range)

• Determine dissociation constants through global fitting to a Langmuir 1:1 model

• For reference, experimental Rmax values of integrins β1 and β5 for ANGPTL4 were 261.1 RU and 229.3 RU, respectively

Affinity Co-precipitation Assay:

• Immobilize His-tagged ANGPTL4 (full-length or specific domains) onto nickel-nitrilotriacetic acid resin

• Incubate with purified matrix proteins at defined concentrations

• Analyze bound and unbound fractions by immunoblotting

• Include appropriate controls with resin treated with buffer only

These methodologies have successfully demonstrated that ANGPTL4 interacts with vitronectin and fibronectin in the wound bed, delaying their proteolytic degradation by metalloproteinases .

How can I distinguish between different forms of ANGPTL4 (full-length vs. cleaved) in experimental samples?

ANGPTL4 exists in multiple forms, including full-length protein and cleaved fragments with distinct biological activities. To distinguish between these forms:

• Western blotting with domain-specific antibodies:

  • Use antibodies targeting the N-terminal domain (~25 kDa after cleavage)

  • Use antibodies targeting the C-terminal domain (~45 kDa for full-length, ~20 kDa for C-terminal fragment)

  • The choice of reducing vs. non-reducing conditions affects band patterns due to disulfide bonds

• Functional assays:

  • The N-terminal fragment has higher activity in LPL inactivation than the uncleaved protein

  • C-terminal fragments have distinct roles in angiogenesis regulation

• Recombinant protein controls:

  • Include purified recombinant domains (nANGPTL4 or cANGPTL4) as size and specificity controls

The ability to distinguish these forms is critical as they possess different functional properties - the N-terminal domain primarily mediates LPL inactivation and affects triglyceride clearance, while the C-terminal domain interacts with integrins and affects cell-matrix communication .

What is the optimal protocol for analyzing ANGPTL4 interactions with integrins using fluorescence-based techniques?

ANGPTL4 has been shown to interact with integrins β1 and β5 to modulate keratinocyte migration during wound healing . For fluorescence-based analysis of these interactions:

• Flow cytometry with FITC-conjugated antibodies:

  • Recommended antibody concentration: 0.25 μg per 10^6 cells

  • For FITC-conjugated antibodies, proper compensation controls are essential to account for spectral overlap

  • Store antibody at -20°C to -70°C; avoid repeated freeze-thaw cycles

• Immunofluorescence co-localization:

  • Use FITC-conjugated anti-ANGPTL4 antibodies in combination with differently labeled anti-integrin antibodies (e.g., Texas Red, Cy5)

  • Include controls for autofluorescence and non-specific binding

  • Analyze using confocal microscopy with appropriate filter sets

• FRET (Förster Resonance Energy Transfer):

  • For direct protein-protein interaction studies, consider using FITC as donor fluorophore and a compatible acceptor fluorophore on the integrin antibody

  • This technique can provide evidence of molecular proximity (<10 nm)

When using these methods, it's important to note that the storage buffer for FITC-conjugated antibodies typically contains 50% glycerol and 0.03% Proclin 300 as preservative , which should be considered when designing experiments.

What methodological approaches are most effective for studying ANGPTL4's role in inflammatory responses?

Research has implicated ANGPTL4 in regulating inflammatory processes, particularly in colonic inflammation and liver transplantation. The following methodological approaches have proven effective:

• In vivo models with genetic modification:

  • ANGPTL4-deficient mice (ANGPTL4^-/-) exhibit exacerbated colonic inflammation in DSS or stearic acid models

  • Bone marrow transplantation experiments help distinguish the intrinsic role of colonic ANGPTL4 in regulating leukocyte infiltration

• Cytokine profiling:

  • Measure pro-inflammatory cytokines like IL-6, IL-1β, and TNF-α

  • Compare cytokine profiles between ANGPTL4-sufficient and deficient models

  • Use both protein (ELISA) and mRNA (qPCR) measurements for comprehensive assessment

• Gene expression analysis:

  • Microarray gene expression profiling has revealed that DSS-treated ANGPTL4^-/- mice show enrichment for genes involved in leukocyte migration and infiltration

  • Expression profiles from ANGPTL4^-/- mice show close association to inflamed ulcerative colitis patterns

• Mechanistic studies:

  • Investigate ANGPTL4-mediated regulation of tristetraprolin expression through CREB and NF-κB transcription factors

  • Examine the effect on chemokine stability

These approaches have demonstrated that ANGPTL4 protects against acute colonic inflammation, and its absence exacerbates inflammation severity .

How can I effectively study ANGPTL4's role in liver transplantation and acute rejection models?

ANGPTL4 has emerged as an important regulator in liver transplantation outcomes, particularly in relation to acute rejection (AR). To study this role effectively:

• Animal transplantation models:

  • Rat orthotopic liver transplantation models have shown reduced ANGPTL4 expression during AR, while increased ANGPTL4 levels are associated with immune tolerance

  • Administration of ANGPTL4 recombinant protein improved liver function and suppressed inflammation in these models

• Kupffer cell polarization analysis:

  • Examine M1 versus M2 polarization states of Kupffer cells (KCs) in the presence/absence of ANGPTL4

  • Flow cytometry with appropriate markers can quantify KC polarization states

  • FITC-conjugated anti-ANGPTL4 antibodies can be used to track ANGPTL4 association with KCs

• Co-culture experimental designs:

  • Establish co-cultures of hepatocytes and KCs to study cellular interactions

  • Examine how hepatocyte-derived ANGPTL4 modulates KC polarization

  • Investigate the involvement of the NF-κB signaling pathway

Research has demonstrated that ANGPTL4 promotes M2 polarization of KCs and improves outcomes in liver transplantation models .

What are the key methodological considerations for investigating ANGPTL4's role in renal fibrosis?

Recent research has identified ANGPTL4 as a key fibrogenic molecule in diabetic kidney disease. When investigating this role:

• Tissue-specific expression models:

  • Both podocyte-specific and tubule-specific ANGPTL4 expression contribute to fibrogenic phenotypes

  • Use appropriate cell-type specific promoters for genetic manipulation

• Temporal expression analysis:

  • Time-dependent emergence of ANGPTL4 expression has been observed in diabetic (fibrotic) kidneys

  • Design sampling timepoints accordingly to capture expression dynamics

• Mechanistic pathway investigation:

  • Study the interaction between podocyte/tubule-secreted ANGPTL4 and Integrin-β1

  • Examine how this interaction influences the association between dipeptidyl-4 with Integrin-β1

  • Investigate the heterodimerization of TGFβR1 and TGFβR2 and subsequent Smad3 phosphorylation

• Metabolic parameter assessment:

  • Evaluate the impact of ANGPTL4 on genes involved in fatty acid oxidation

  • Measure levels of pro-inflammatory cytokines, epithelial-to-mesenchymal transition markers, and endothelial-to-mesenchymal transition markers

These approaches have revealed that targeted inhibition of kidney-specific ANGPTL4 may be a promising therapeutic strategy for managing diabetic kidney disease .

What are the optimal storage and handling conditions for FITC-conjugated ANGPTL4 antibodies?

To maintain the integrity and performance of FITC-conjugated ANGPTL4 antibodies:

• Storage conditions:

  • Store at -20°C to -70°C for long-term preservation

  • For short-term (1 month), store at 2-8°C under sterile conditions after reconstitution

  • For medium-term (6 months), store at -20°C to -70°C under sterile conditions after reconstitution

  • Avoid repeated freeze-thaw cycles to prevent degradation of both antibody and fluorophore

• Reconstitution protocols:

  • Typically reconstitute lyophilized antibodies at 0.5 mg/mL in sterile PBS

  • Some preparations contain 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative

• Light sensitivity considerations:

  • FITC is highly sensitive to photobleaching

  • Minimize exposure to light during all handling steps

  • Store in amber tubes or wrapped in aluminum foil

  • Work in reduced ambient lighting when possible

• Stability assessments:

  • If stored properly, FITC-conjugated antibodies typically maintain activity for 12 months from date of receipt

  • Consider including positive controls with each experiment to verify continued antibody performance

These handling precautions ensure optimal performance in fluorescence-based applications including flow cytometry, immunofluorescence microscopy, and other visualization techniques.

How should I interpret contradictory findings when studying ANGPTL4 in different disease models?

ANGPTL4 exhibits context-dependent functions that can appear contradictory across different experimental systems. When facing seemingly conflicting data:

• Consider tissue-specific effects:

  • ANGPTL4 functions differently in adipose tissue, liver, kidney, and intestine

  • In liver transplantation, ANGPTL4 promotes M2 macrophage polarization (anti-inflammatory)

  • In colonic inflammation, ANGPTL4 protects against inflammatory damage

  • In kidney disease, ANGPTL4 has been identified as a fibrogenic molecule

• Distinguish between systemic and local effects:

  • Circulating ANGPTL4 primarily affects lipid metabolism through LPL inhibition

  • Locally produced ANGPTL4 may have distinct paracrine effects on tissue remodeling and inflammation

• Account for full-length versus cleaved fragments:

  • The N-terminal domain has higher activity in LPL inactivation

  • The C-terminal domain interacts with integrins and affects cell migration

  • Research findings may differ depending on which form predominates in the experimental system

• Experimental design variables:

  • Acute versus chronic disease models yield different results

  • The microenvironment (e.g., presence of matrix proteins, inflammatory mediators) affects ANGPTL4 function

  • Compensatory mechanisms may emerge in genetic knockout models but not in acute interventions

Understanding these variables will help reconcile apparently contradictory findings and develop a more nuanced understanding of ANGPTL4's multifaceted biological roles.