crfb12 Antibody

What is crfb12 and what role does it play in zebrafish biology?

crfb12 (Cytokine receptor family member B12) is a transmembrane protein expressed in zebrafish (Danio rerio) that functions within cytokine signaling pathways. It belongs to the cytokine receptor family with a UniProtID of Q1RM05 . The receptor participates in immune signaling cascades similar to other cytokine receptors, potentially playing roles in development, immune function, and stress responses.

Methodologically, when studying crfb12:

• Use whole-mount in situ hybridization to visualize expression patterns during development

• Employ RT-PCR or qPCR to quantify transcript levels across different tissues

• Analyze protein expression through immunohistochemistry with specific anti-crfb12 antibodies

• Consider knock-down or knockout approaches using morpholinos or CRISPR-Cas9 to assess functional roles

What applications are most appropriate for crfb12 antibodies in zebrafish research?

Based on available data, crfb12 antibodies have been validated for several research applications:

For optimal results, researchers should:

• Perform antibody validation in their specific experimental context

• Include appropriate positive and negative controls

• Test multiple antibody dilutions to determine optimal concentration

• Consider using biotin-conjugated variants for signal amplification in applications with low target abundance

How do different conjugates of crfb12 antibody influence experimental outcomes?

Different conjugates of crfb12 antibodies offer distinct advantages depending on the experimental application:

When designing experiments with conjugated crfb12 antibodies:

• Consider tissue autofluorescence when using FITC-conjugated antibodies

• For FITC applications, protect from light and use appropriate mounting media to prevent photobleaching

• For biotin conjugates, block endogenous biotin in zebrafish tissues to reduce background

• Validate each conjugate independently for your specific application

What are the optimal fixation and permeabilization protocols for crfb12 immunohistochemistry in zebrafish embryos?

Successful detection of crfb12 in zebrafish embryos requires careful optimization of fixation and permeabilization:

Recommended Protocol:

• Fix embryos in 4% paraformaldehyde (PFA) in PBS for 2-4 hours at room temperature or overnight at 4°C

• Wash 3× in PBS-T (PBS with 0.1% Tween-20)

• For embryos >24 hpf, permeabilize with proteinase K (10 μg/mL) for age-appropriate duration:

  • 24-48 hpf: 5-10 minutes

  • 48-72 hpf: 15-20 minutes

  • 72 hpf: 20-30 minutes

• Re-fix briefly (20 minutes) in 4% PFA

• Block with 10% normal goat serum in PBS-T for 1-2 hours

• Incubate with crfb12 antibody (typically 1:100-1:500 dilution) overnight at 4°C

Key Optimization Considerations:

• Excessive fixation can mask epitopes; consider antigen retrieval if signal is weak

• The milli fluidic device approach demonstrated by recent research can improve consistency of whole-mount antibody staining while reducing washing time by approximately 50%

• For co-localization studies, ensure compatibility of fixation methods for all target epitopes

How can crfb12 antibody be incorporated into zebrafish models of immune response?

crfb12 antibodies can provide valuable insights into zebrafish immune response models:

Methodological Approaches:

• Infection models: Monitor crfb12 expression changes following bacterial or viral challenge

• Inflammation studies: Assess crfb12 localization in relation to inflammatory markers

• Developmental immunology: Track crfb12 expression during immune system development

• Stress response: Evaluate crfb12 in relation to HPI axis activation

When designing such experiments:

• Include appropriate time points (early and late responses)

• Consider tissue-specific responses (whole embryo vs. specific organs)

• Use multiple detection methods (protein and transcript level analysis)

• Correlate with functional readouts of immune response

Zebrafish offer unique advantages for studying cytokine receptor functions, including optical transparency, rapid development, and conservation of immune pathways with mammals.

What controls should be included when performing crfb12 antibody validation?

Rigorous antibody validation is essential for reliable research outcomes:

Essential Controls for crfb12 Antibody Validation:

• Negative controls:

  • Primary antibody omission

  • Incubation with non-immune IgG of same species and concentration

  • Blocking peptide competition (using the immunizing peptide sequence from zebrafish cytokine receptor family member B12 protein (106-130AA))

  • Tissue from crfb12 knockdown/knockout models if available

• Positive controls:

  • Tissues known to express crfb12 (based on transcript data)

  • Overexpression systems (in vitro or in vivo)

  • Western blot showing band of appropriate molecular weight

• Technical validation:

  • Multiple antibody dilutions to establish optimal concentration

  • Testing different fixation and permeabilization methods

  • Comparison of different antibody lots

  • Cross-validation with orthogonal methods (e.g., in situ hybridization)

Document all validation steps methodically to ensure reproducibility and reliability of results.

How can crfb12 antibody be integrated with advanced imaging techniques for zebrafish research?

Advanced imaging approaches can maximize information gained from crfb12 antibody staining:

Methodological Integration Strategies:

• Lightsheet microscopy:

  • Enables whole-embryo imaging with minimal phototoxicity

  • Optimize clearing methods compatible with crfb12 antibody (e.g., CLARITY, CUBIC)

  • Maintain sample in appropriate mounting medium that preserves fluorophore stability

• Super-resolution microscopy:

  • For sub-cellular localization of crfb12

  • Consider photo-switchable fluorophore conjugates for STORM/PALM

  • Optimize sample preparation to minimize background and maximize signal-to-noise ratio

• Intravital imaging:

  • For real-time dynamics in transgenic reporter lines

  • Can be combined with photo-convertible proteins to track cell movement

  • Correlate with crfb12 antibody staining in fixed samples

• Automated high-content imaging:

  • The milli fluidic device approach allows standardized orientation of embryos for consistent imaging planes

  • Enables higher throughput analysis across multiple experimental conditions

Ensure consistent imaging parameters across experimental groups for valid quantitative comparisons.

What methodological approaches can resolve contradictory data when studying crfb12 function?

When facing conflicting results regarding crfb12 function, systematic troubleshooting is essential:

Resolution Framework:

• Antibody validation reassessment:

  • Verify antibody specificity using multiple approaches

  • Test alternative antibody lots or sources

  • Confirm epitope accessibility in your experimental conditions

• Technical variables analysis:

  • Create a comprehensive table documenting all experimental variables:

  Variable	Experiment A	Experiment B	Potential Impact
  Antibody concentration	1:200	1:500	Signal intensity
  Fixation duration	2 hours	Overnight	Epitope masking
  Development stage	24 hpf	48 hpf	Expression differences
  Detection method	Chromogenic	Fluorescent	Sensitivity variance
  Genetic background	AB strain	TU strain	Genetic modifiers

• Multi-method verification:

  • Supplement antibody detection with transcript analysis (qPCR, in situ)

  • Employ genetic approaches (morpholinos, CRISPR) to confirm function

  • Use multiple antibodies targeting different epitopes if available

• Biological context consideration:

  • Evaluate environmental variables (temperature, stress)

  • Consider developmental timing precision

  • Assess potential compensatory mechanisms

How does crfb12 signaling in zebrafish compare to cytokine signaling in mammalian systems?

Understanding comparative aspects of cytokine receptor signaling enhances translational relevance:

Comparative Analysis Framework:

• Sequence homology analysis:

  • Perform phylogenetic analysis of crfb12 across species

  • Identify conserved functional domains and signaling motifs

  • Map epitope regions targeted by antibodies relative to conserved domains

• Signaling pathway conservation:

  • Investigate downstream pathway components (similar to how CRF pathways involve the HPI axis in zebrafish, analogous to the HPA axis in mammals)

  • Compare expression patterns during development and in response to stimuli

  • Assess functional conservation through rescue experiments

• Cross-reactivity testing:

  • While most crfb12 antibodies are zebrafish-specific, testing for cross-reactivity with mammalian homologs can provide insight into structural conservation

  • Examine potential for cross-species complementation experiments

  • Consider domain-specific antibodies for comparative studies

The zebrafish HPI axis, governed by various signaling molecules including cytokine receptors, shares significant functional homology with mammalian stress response systems, despite some structural differences .

What innovative applications of crfb12 antibodies are emerging in zebrafish research?

Cutting-edge applications for crfb12 antibodies include:

Emerging Methodological Approaches:

• High-throughput screening platforms:

  • Integration with automated imaging and analysis pipelines

  • Application in drug discovery for immune modulators

  • Combined with CRISPR screens for pathway component identification

• Single-cell analysis:

  • Antibody-based cell sorting for transcriptomic profiling

  • Mass cytometry (CyTOF) with metal-conjugated antibodies for multi-parameter analysis

  • Spatial transcriptomics correlated with protein localization

• Microfluidic applications:

  • The recently developed multi-depth spiral milli fluidic device for whole mount zebrafish antibody staining represents a significant methodological advancement

  • Enables consistent embryo orientation and reduces processing time by approximately 50%

  • Provides standardized conditions for comparative studies

• Biosensor development:

  • Antibody-based detection of signaling pathway activation

  • In vivo reporters for cytokine receptor activity

  • Development of activity-sensing nanobodies for live imaging

These approaches are expanding our ability to understand cytokine receptor biology in zebrafish models with increased precision, throughput, and complexity.

What are the key considerations for using crfb12 antibodies in whole-mount vs. sectioned zebrafish samples?

The choice between whole-mount and sectioned samples significantly impacts experimental design:

Optimization Strategies:

• For whole-mount: extend antibody incubation times (24-72 hours), use higher antibody concentrations, optimize detergent concentration

• For sections: optimize section thickness (typically 10-20 μm), reduce antibody concentration, shorter incubation times

• Consider clearing techniques (CLARITY, CUBIC, etc.) for improved signal-to-noise in whole-mount samples

• The milli fluidic device approach can significantly improve consistency and reduce processing time for whole-mount staining

How can crfb12 antibodies be used to investigate stress response pathways in zebrafish models?

Zebrafish stress response provides a valuable comparative model for understanding cytokine signaling:

Experimental Design Framework:

• Stress induction protocols:

  • Acute stressors: handling, temperature shift, hypoxia

  • Chronic stressors: social isolation, unpredictable chronic stress

  • Chemical stressors: cortisol exposure, CRF administration

• Analysis timepoints:

  • Immediate response (0-1 hours)

  • Early response (1-6 hours)

  • Late response (6-24 hours)

  • Chronic adaptation (days-weeks)

• Multilevel assessment:

  • Transcript analysis of crfb12 and related pathway components

  • Protein expression/localization using crfb12 antibodies

  • Correlation with cortisol levels and other stress markers

  • Behavioral phenotyping

• Key pathway interactions:

  • Examine relationship between crfb12 and HPI axis components

  • Similar to how maternal cortisol mediates HPI axis development in zebrafish

  • Assess potential interaction with CRF pathways, which are critical for zebrafish camouflage behavior