nr2f1b Antibody

Basic Research Questions

• What is NR2F1b and why is it significant for vascular research?

  NR2F1b is a transcription factor that plays a critical role in blood vessel formation in zebrafish. Research demonstrates that NR2F1b regulates venous specification and angiogenic patterning. Loss of nr2f1b results in decreased vein cell numbers and reduced expression of vein-specific markers including flt4 and mrc1. NR2F1b has been shown to affect intersegmental vessel (ISV) cell numbers and growth by influencing angiogenic cell migration and/or proliferation .

  Morpholino knockdown of nr2f1b leads to pericardial edema and circulation defects, while overexpression under the fli promoter increases venous and ISV endothelial cell numbers, indicating its necessary role in vascular development .

• How does NR2F1b relate to other NR2F family members?

  NR2F1b is related to but functionally distinct from other NR2F family members:

  Family Member	Main Functions	Model Systems	Sequence Similarity to nr2f1b
  nr2f1a (zebrafish)	Not fully characterized	Zebrafish	52% identity with nr2f1b morpholino target sequence
  nr2f2 (zebrafish)	Minor role in venous identity, critical for lymphangiogenesis	Zebrafish	28% identity with nr2f1b morpholino target sequence
  NR2F1 (mammalian)	CNS and peripheral nervous system development, inner ear cell differentiation, tumor dormancy regulation	Mouse, Human	Zebrafish nr2f1b ortholog
  NR2F2 (mammalian)	Major regulator of venous identity, angiogenic growth, lymphatic endothelial fate determination	Mouse, Human	Functionally similar to zebrafish nr2f1b

  Understanding these relationships is crucial when selecting antibodies to ensure specificity for the intended target .

• What experimental applications are suitable for NR2F1b antibodies?

  Based on the research data from related NR2F family antibodies, NR2F1b antibodies can be used in:

  • Immunohistochemistry (IHC-P): For detection in paraffin-embedded tissue sections

  • Immunocytochemistry/Immunofluorescence (ICC/IF): For cellular localization studies

  • Western Blot (WB): For protein expression analysis

  • Immunoprecipitation (IP): For protein-protein interaction studies

  • ChIP/CUT&RUN-seq: For studying DNA binding and transcriptional regulation

  When selecting an antibody for a specific application, validation data should be consulted to ensure compatibility with the intended use and target species .

Advanced Research Questions

• How can I validate the specificity of an NR2F1b antibody?

  Rigorous validation is essential due to the high sequence homology between NR2F family members. A comprehensive validation approach should include:

  • RT-PCR verification: After morpholino knockdown, confirm specificity by testing effects on nr2f1b, nr2f1a, and nr2f2 levels. Specific morpholinos should decrease only the target transcript .

  • Rescue experiments: Overexpression of nr2f1b in morphants should rescue the phenotype if the antibody is specific .

  • CRISPR knockout controls: Use CRISPR/Cas9-edited cell lines lacking NR2F1b as negative controls for antibody testing .

  • Cross-reactivity testing: Test against recombinant NR2F1 and NR2F2 proteins to ensure specificity .

  • Multiple antibody comparison: Use different antibody clones targeting distinct epitopes to confirm consistent results .

  For example, research on nr2f1b morpholino specificity showed that while the morpholino had 52% and 28% sequence identity with nr2f1a and nr2f2 respectively, it specifically decreased nr2f1b levels without affecting nr2f1a or nr2f2 .

• What are the optimal conditions for detecting NR2F1b in zebrafish vascular tissue?

  Based on experimental protocols used in nr2f1b research:

  1. Fixation: Paraformaldehyde fixation (typically 4%) is recommended for preserving tissue architecture while maintaining epitope accessibility.

  2. Antibody concentration: Titrate antibody concentrations carefully. For related NR2F antibodies, concentrations between 0.4-2 μg/ml have been tested, with 1 μg/ml often providing optimal results balancing specific nuclear signal and reduction of background .

  3. Antigen retrieval: For paraffin-embedded tissues, heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 is recommended before commencing with immunostaining .

  4. Controls: Include appropriate negative controls:

     • Primary antibody only

     • Secondary antibody only

     • No antibody (DAPI only)

  5. Detection method: For fluorescent detection in zebrafish embryos, use confocal microscopy to visualize vascular structures. Transgenic lines expressing fluorescent proteins in endothelial cells (such as Tg(fli1:EGFP)) can be used to co-localize NR2F1b with vascular structures .

• How can I interpret conflicting nuclear versus nucleolar NR2F1 localization data?

  Contradictory observations regarding NR2F1 localization require careful experimental design and interpretation:

  1. Antibody selection: Multiple studies have identified that certain antibodies (particularly the monoclonal Ab clone H8132) may show nucleolar-like aggregates that appear to be artifacts rather than true biological localization .

  2. Validation approach: Use multiple antibodies targeting different epitopes of NR2F1b and compare localization patterns.

  3. Controls: Include both wildtype and NR2F1-null samples to confirm specificity.

  4. Fixation effects: Test different fixation methods as they can affect nuclear protein localization patterns.

  5. Expression level considerations: Nucleolar-like staining may be concentration-dependent and vary with expression levels of NR2F1 .

  Research has shown that nucleolar localization of NR2F1 observed with some antibodies is likely artificial and does not represent functional localization. The diffuse nuclear staining pattern is more consistent with its role as a transcription factor .

• What approaches can help differentiate between NR2F1b and related family members in experimental settings?

  Given the structural similarity between NR2F family members, several approaches can help ensure specificity:

  1. Epitope selection: Choose antibodies targeting non-conserved regions between family members.

  2. Isoform-specific RT-PCR: Use primers spanning exon-exon junctions unique to nr2f1b to validate knockdown or expression changes at the transcript level .

  3. Genetic models: Utilize genetic knockdown/knockout models specific for nr2f1b:

     • Morpholino targeting i1e2 junction of nr2f1b demonstrated specificity with minimal effects on nr2f1a and nr2f2

     • CRISPR/Cas9 knockout models can provide definitive specificity controls

  4. Functional validation: Complement antibody studies with functional assays, as nr2f1b has distinct phenotypic effects from nr2f2 in zebrafish vascular development .

  For example, in one study, RT-PCR confirmed that nr2f1b morpholino specifically decreased nr2f1b transcript levels without affecting nr2f1a or nr2f2 expression, validating the specificity of their approach .

• How can I use NR2F1b antibodies to study its interaction with the Notch signaling pathway?

  To investigate NR2F1b-Notch signaling interactions, consider these methodological approaches:

  1. Co-immunoprecipitation: Use validated NR2F1b antibodies to immunoprecipitate protein complexes, followed by Western blot analysis for Notch pathway components.

  2. Proximity ligation assay: Detect protein-protein interactions between NR2F1b and Notch pathway components with high spatial resolution.

  3. Combined genetic approaches:

     • Treat embryos with DAPT (γ-secretase inhibitor) to inhibit Notch activity and assess changes in NR2F1b levels and localization

     • In rbpsuh (RBP-J) morphants (where Notch signaling is impaired), nr2f1b expression increases, suggesting negative regulation by Notch activity

  4. Chromatin immunoprecipitation (ChIP): Use NR2F1b antibodies for ChIP followed by sequencing to identify genomic binding sites that may overlap with Notch signaling targets.

  Research has shown that nr2f1b expression increases in both rbpsuh morphants and DAPT-treated embryos, suggesting that nr2f1b is negatively regulated by Notch activity .

• What methodologies are effective for studying NR2F1b in tumor dormancy research?

  While NR2F1 has been identified as a master regulator of tumor cell dormancy, specific approaches for studying NR2F1b in this context include:

  1. Quantification methods:

     • For microscopy-based quantification, use a stringent mask for strong nuclear NR2F1 signal, which appears as prominent clusters

     • Mean fluorescence intensity (MFI) of nuclear NR2F1 can be measured in 3D cultures to assess expression levels

  2. 3D culture systems: Plate cells at low density in Matrigel and treat with compounds of interest for several days before immunostaining for NR2F1b .

  3. In vivo models:

     • Use chicken chorioallantoic membrane (CAM) assays with human tumor cells

     • Treat tumors with compounds of interest and then isolate and immunostain cells (using vimentin to distinguish human tumor cells)

  4. Expression analysis: Analyze NR2F1b mRNA using RT-PCR and protein using Western blot and immunofluorescence to correlate expression with dormancy phenotypes .

  Research showed that NR2F1-positive cells increased from approximately 4% in control tumors to 42% in treated tumors, demonstrating significant upregulation during dormancy induction .

• How can I use NR2F1b antibodies to identify novel downstream vascular genes?

  To identify novel vascular genes regulated by NR2F1b:

  1. Knockdown approach: Use morpholinos targeting nr2f1b and/or related factors (such as isl2) to manipulate expression .

  2. Transcriptomic analysis:

     • Use microarray or RNA-seq following nr2f1b knockdown

     • Apply statistical analysis using linear models and empirical Bayesian adjustment to identify differentially expressed genes

     • Control for false discovery rate using the Benjamini and Hochberg method

     • Consider genes with expression fold change ≥2.5 or <0.5 as significantly affected

  3. Validation of targets:

     • Use nr2f1b antibodies in ChIP-seq to identify direct binding targets

     • Perform RT-PCR and Western blot to confirm expression changes

     • Use in situ hybridization to examine spatial expression patterns of candidate genes

  4. Functional characterization:

     • Conduct loss-of-function studies of identified targets and assess vascular phenotypes

     • Use rescue experiments to establish genetic hierarchy

  Research using this approach identified several genes regulated by nr2f1b, including coagulation factor II (f2), cdkal1, aqp12, and sesn3, with fold changes ranging from 3.7 to 5.1 following nr2f1b knockdown .

Methodology and Technical Considerations

• What are the best practices for developing and validating new NR2F1b antibodies?

  Based on recent antibody development approaches, consider these methodological steps:

  1. Epitope selection:

     • Target unique regions with low homology to related proteins

     • Consider both N-terminal and C-terminal epitopes for validation

  2. Recombinant antibody development:

     • Clone immunoglobulin G (IgG) variable domains from hybridomas

     • Engineer plasmid backbones allowing for IgG subclass switching without altering target binding specificity

     • Express in appropriate systems (mammalian cells like COS-1)

  3. Validation pipeline:

     • Test in multiple applications (WB, IF-ICC, IHC)

     • Include positive and negative controls (knockouts, competing antigens)

     • Assess cross-reactivity with related proteins (NR2F1a, NR2F2)

     • Sequence validation to confirm uniqueness

  4. Modern approaches:

     • Consider computational antibody design using fine-tuned RFdiffusion networks for epitope-specific binding

     • Validate designed antibodies using multiple orthogonal biophysical methods including cryo-EM

  For example, a systematic approach for recombinant monoclonal antibody validation included testing multiple clones in transiently transfected cells using immunofluorescence, with subsequent DNA sequencing of positive clones to confirm specificity .

• What controls are essential when using NR2F1b antibodies in various experimental applications?

  Rigorous controls are crucial for reliable NR2F1b antibody experiments:

  1. Western Blot controls:

     • Positive control: Tissue/cells known to express NR2F1b

     • Negative control: NR2F1b knockout/knockdown samples

     • Loading control: β-actin or GAPDH

     • Specificity control: Pre-incubation with blocking peptide

  2. Immunohistochemistry/Immunofluorescence controls:

     • Primary antibody omission: Secondary antibody only

     • Isotype control: Non-specific IgG of same isotype

     • Absorption control: Pre-incubation with antigen

     • Positive tissue control: Known NR2F1b-expressing tissue

     • Negative tissue control: Tissue from knockout models

  3. IP controls:

     • Input sample (pre-IP)

     • Non-specific IgG IP

     • Validation with secondary detection method

  4. Multiple antibody validation:

     • Use multiple antibodies targeting different epitopes

     • Compare staining patterns and expression levels

     • Test different fixation and antigen retrieval methods

  For example, a study examining NR2F1 localization used multiple controls including primary antibody only, secondary antibody only, and DAPI only to distinguish true signal from background and autofluorescence .

• How can I troubleshoot inconsistent results with NR2F1b antibodies?

  When facing inconsistent results, consider these methodological approaches:

  1. Antibody quality issues:

     • Test multiple lots of the same antibody

     • Compare with antibodies from different suppliers/clones

     • Validate with genetic approaches (siRNA, CRISPR knockout)

  2. Technical variables to adjust:

     • Fixation optimization: Test multiple fixation methods and durations

     • Antibody concentration: Titrate across a range (e.g., 0.4-2 μg/ml)

     • Incubation conditions: Vary temperature, duration, buffer composition

     • Antigen retrieval: Test different methods (heat, enzymatic) and buffers

     • Blocking conditions: Test different blocking agents and concentrations

  3. Sample-specific considerations:

     • Expression level variations between tissues/developmental stages

     • Post-translational modifications affecting epitope recognition

     • Protein-protein interactions masking epitopes

  4. Biological validation:

     • Correlate antibody results with mRNA expression

     • Use reporter constructs or tagged proteins as independent validation

  In one study investigating NR2F1 localization, researchers found that adjusting antibody concentration significantly affected the pattern of staining, with different concentrations revealing either diffuse nuclear signal or predominantly nuclear aggregates .

• What specialized techniques can be used to study NR2F1b function in vascular development?

  Advanced methodological approaches for investigating NR2F1b function include:

  1. Zebrafish-specific techniques:

     • Morpholino knockdown: Use splice-blocking morpholinos targeting intron-exon junctions (e.g., i1e2 junction)

     • Transgenic overexpression: Use the fli1 promoter to drive endothelial-specific expression

     • Time-lapse imaging: Monitor ISV growth and cell migration in real-time

     • CRISPR/Cas9 gene editing: Generate stable genetic knockouts

  2. Vascular phenotyping methods:

     • Vascular cell counting: Quantify venous cells and ISV endothelial cells

     • Marker expression analysis: Examine expression of vein-specific markers (flt4, mrc1)

     • Functional circulatory assessment: Evaluate pericardial edema and blood flow

  3. Signaling pathway integration:

     • Pharmacological manipulation: Use DAPT to inhibit Notch signaling and assess effects on NR2F1b

     • Genetic interaction studies: Combine nr2f1b knockdown with manipulation of other pathways

     • Rescue experiments: Test the ability of downstream factors to rescue nr2f1b knockdown phenotypes

  Research demonstrated that nr2f1b morphants showed reduced ISV cell numbers and impaired ISV growth, while overexpression of nr2f1b under the fli promoter increased both venous and ISV endothelial cell numbers, providing functional validation of its role in vascular development .

• How do I design experiments to investigate NR2F1b's transcriptional targets?

  To identify and characterize NR2F1b transcriptional targets:

  1. Genome-wide approaches:

     • ChIP-seq: Use validated NR2F1b antibodies to immunoprecipitate chromatin, followed by sequencing to identify genome-wide binding sites

     • CUT&RUN-seq: Alternative to ChIP with improved signal-to-noise ratio for transcription factor binding site identification

     • RNA-seq: Compare transcriptomes between wildtype and nr2f1b knockdown/knockout models

     • ATAC-seq: Identify changes in chromatin accessibility upon NR2F1b manipulation

  2. Target validation methods:

     • Reporter assays: Test direct regulation using luciferase reporters containing candidate target promoters/enhancers

     • Site-directed mutagenesis: Mutate predicted NR2F1b binding sites in regulatory regions

     • Electrophoretic mobility shift assay (EMSA): Confirm direct binding to specific DNA sequences

  3. Functional characterization:

     • Gene knockdown: Test the effects of silencing candidate targets on vascular development

     • Epistasis experiments: Determine genetic hierarchy through combined knockdown/overexpression

     • In vivo validation: Examine expression patterns of targets in zebrafish vascular tissues

  Research using gene expression analysis following nr2f1b knockdown identified several potential targets, with fold changes ranging from 3.37 to 5.11, including genes involved in vascular development and regulation .