crfb12 Antibody, FITC conjugated

What is crfb12 and why is it significant in zebrafish research?

Cytokine receptor family member B12 (crfb12) is a membrane protein involved in immunological signaling pathways in zebrafish. This receptor is particularly important for studying immune system development and function in the zebrafish model organism. Zebrafish (Danio rerio) provide an excellent vertebrate model for studying immune system development due to their optical transparency during early development and genetic tractability .

Researchers typically use crfb12 antibodies to:

• Track expression patterns during zebrafish development

• Examine immune cell populations in various experimental conditions

• Study cytokine signaling pathways in normal and disease states

The FITC-conjugated version enables direct visualization without requiring secondary antibody labeling, making it particularly valuable for flow cytometry applications.

How does FITC conjugation to antibodies actually work?

FITC (fluorescein isothiocyanate) conjugation involves a chemical reaction between the isothiocyanate group of FITC and primary amines (lysine residues) on the antibody. This creates a stable thiourea linkage . The process typically involves:

• Preparation of the antibody in an alkaline buffer (typically pH 9.2) to increase reactivity of lysine residues

• Addition of FITC dissolved in an anhydrous organic solvent (typically DMSO)

• Incubation (typically 2 hours at room temperature)

• Removal of unbound FITC through dialysis or gel filtration

For optimal labeling, reaction conditions must be carefully controlled. The maximal molecular fluorescein/protein (F/P) ratio is achieved when reaction temperature, pH, and protein concentration are high. Specifically, optimal conjugation typically occurs at room temperature, pH 9.5, and an initial protein concentration of 25 mg/ml for 30-60 minutes .

What are the recommended storage conditions for crfb12 Antibody, FITC conjugated?

Based on manufacturer recommendations for crfb12 Antibody, FITC conjugated:

• Upon receipt, store at -20°C or -80°C

• Avoid repeated freeze-thaw cycles

• The antibody is typically supplied in a buffer containing:

  • 50% Glycerol

  • 0.01M PBS, pH 7.4

  • 0.03% Proclin 300 as a preservative

For short-term use (less than a month), the conjugated antibody can be stored at 4°C protected from light. FITC conjugates are particularly sensitive to photobleaching, so protection from light during storage and handling is essential .

How can I determine the optimal concentration of crfb12 Antibody, FITC conjugated for my experiments?

Determining the optimal concentration requires a systematic titration approach:

• Prepare a series of antibody dilutions (typically 2-fold dilutions ranging from 1:10 to 1:1280)

• Test each dilution in your experimental system (flow cytometry, ELISA, etc.)

• Analyze the signal-to-noise ratio to identify the dilution that provides maximum specific signal with minimal background

For flow cytometry applications, optimal titration can be determined by:

• Plotting the staining index (mean fluorescence intensity of positive population minus mean fluorescence intensity of negative population, divided by twice the standard deviation of the negative population)

• The antibody concentration giving the highest staining index represents the optimal titer

In general, you should perform this titration experiment in conditions closely matching your final experimental setup, as the optimal concentration may vary depending on cell type, fixation method, and other experimental variables .

What methods can I use to validate the specificity of crfb12 Antibody, FITC conjugated?

Several approaches can be used to validate antibody specificity:

• Positive and negative controls:

  • Test the antibody on cell populations known to express crfb12 (positive control)

  • Test on cell populations that do not express crfb12 (negative control)

  • For zebrafish studies, testing on appropriate immune cell populations versus non-immune tissues is recommended

• Blocking experiments:

  • Pre-incubate the antibody with the immunizing peptide (for this antibody, the peptide sequence from zebrafish Cytokine receptor family member B12 protein (106-130AA))

  • Compare staining with and without blocking

  • Specific binding should be significantly reduced after blocking

• Knockdown validation:

  • Test the antibody on samples from crfb12 knockdown models (e.g., morpholino-treated embryos)

  • Compare with wildtype samples

  • Signal should be reduced in knockdown samples

• Western blot correlation:

  • Compare flow cytometry results with western blot analysis using the non-conjugated version of the same antibody

  • Correlation between techniques increases confidence in specificity

What considerations are important when designing multicolor panels including crfb12 Antibody, FITC conjugated?

When incorporating FITC-conjugated antibodies into multicolor panels:

• Spectral overlap considerations:

  • FITC emits at approximately 530 nm when excited at 488 nm

  • Be aware of spectral overlap with other fluorophores like PE (phycoerythrin)

  • Proper compensation controls are essential for accurate data interpretation

• Brightness hierarchy:

  • FITC has moderate brightness compared to other fluorophores

  • Reserve FITC for antigens with moderate to high expression

  • For low abundance antigens, consider brighter fluorophores like PE or APC

• Autofluorescence considerations:

  • Zebrafish tissues can exhibit autofluorescence in the FITC channel

  • Include appropriate unstained controls to account for this

  • Consider using spectral unmixing algorithms if available

• Panel design strategy:

  • For a panel including crfb12-FITC, consider pairing with:

    • PE-conjugated antibodies (minimal spectral overlap)

    • APC-conjugated antibodies (minimal spectral overlap)

    • Avoid fluorophores with significant emission in the 530nm range

How can I adapt Single-color Multitarget Flow Cytometry (SM-FC) protocols for use with crfb12 Antibody, FITC conjugated?

SM-FC allows detection of multiple targets using a single fluorochrome by creating graded mean fluorescence intensities (MFIs). For incorporating crfb12 Antibody, FITC conjugated into SM-FC:

• Preparation of dimly labeled antibody:

  • Decrease the volume of crfb12 Antibody, FITC conjugated to create a "dim" label

  • Determine the optimal diluted volume through serial dilution experiments

  • For example, studies have shown that CD3 and CD19 FITC (0.1 and 0.5 µL/test, respectively) yield weakly positive cell populations in SM-FC

• Creating an antibody cocktail:

  • Combine dim crfb12 Antibody, FITC conjugated with another brightly FITC-labeled antibody

  • Ensure there is sufficient separation between the MFIs of the two populations

  • Typical MFI ratios between bright and dim populations should be at least 5-10 fold

• Validation of SM-FC approach:

  • Compare results with conventional multicolor flow cytometry

  • Assess repeatability through multiple analyses of the same samples

  • Previous studies with SM-FC using FITC-conjugated antibodies have achieved CV values of 0.8-5.0%, comparable to multicolor flow cytometry

• Data analysis considerations:

  • Use appropriate gating strategies to identify populations based on fluorescence intensity

  • Include single-stained controls for each antibody to establish appropriate gates

  • Be aware that compensation cannot be performed between antibodies conjugated to the same fluorophore

How does the molecular fluorescein/protein (F/P) ratio affect the performance of FITC-conjugated antibodies?

The F/P ratio is a critical determinant of antibody performance:

What are emerging applications for crfb12 Antibody, FITC conjugated in zebrafish immunology research?

Several cutting-edge applications are emerging for FITC-conjugated antibodies in zebrafish research:

• High-dimensional flow cytometry:

  • Integration with spectral cytometry allows simultaneous detection of more parameters

  • Can be combined with cell sorting for downstream single-cell analysis

  • Enables detailed characterization of immune cell subpopulations during development and disease

• Live imaging applications:

  • FITC-conjugated antibodies can be used for in vivo imaging in zebrafish embryos

  • Particularly valuable due to the optical transparency of zebrafish early developmental stages

  • Can be combined with transgenic reporter lines for multicolor imaging

• Nanoscopy techniques:

  • Super-resolution microscopy approaches can utilize FITC conjugates

  • Provides subcellular localization of crfb12 receptor

  • Allows investigation of receptor clustering and co-localization with signaling partners

• Integrating with single-cell technologies:

  • Flow sorting of crfb12-positive cells for single-cell RNA sequencing

  • Enables correlation of receptor expression with transcriptomic profiles

  • Can reveal novel insights into immune cell heterogeneity and differentiation

What are the most common issues when using FITC-conjugated antibodies and how can they be addressed?

Researchers frequently encounter these challenges with FITC conjugates:

• Photobleaching:

  • FITC is particularly susceptible to photobleaching

  • Solution: Minimize exposure to light during sample preparation and analysis; use antifade reagents in microscopy applications; consider using more photostable conjugates for extended imaging

• pH sensitivity:

  • FITC fluorescence is optimal at alkaline pH and decreases substantially below pH 7

  • Solution: Maintain samples at pH 7.2-8.0 during staining and analysis; buffer systems should be carefully controlled

• High background fluorescence:

  • Can result from non-specific binding or autofluorescence

  • Solution: Include proper blocking reagents; use Fc receptor blocking; include fluorescence-minus-one (FMO) controls; consider background subtraction algorithms

• Inconsistent conjugation results:

  • Variation between conjugation batches can affect experimental consistency

  • Solution: Standardize conjugation protocols; characterize each batch by spectrophotometry; consider using commercial conjugation kits with standardized procedures

How can I optimize protocols for using crfb12 Antibody, FITC conjugated in zebrafish tissue samples?

Zebrafish tissues present unique challenges that require specific optimization:

• Tissue dissociation optimization:

  • Gentle enzymatic dissociation methods (e.g., collagenase, trypsin) preserve surface antigens

  • Mechanical dissociation should be optimized to prevent cell damage

  • Cold PBS with protein (1-2% BSA) helps maintain antigen integrity

• Reducing autofluorescence:

  • Zebrafish tissues, particularly those containing pigment, can have high autofluorescence

  • Pre-treatment with Sudan Black B (0.1-0.5%) can reduce autofluorescence

  • Including unstained controls for each tissue type is essential

• Fixation considerations:

  • Mild fixation (0.5-1% paraformaldehyde for 15-30 minutes) preserves most epitopes

  • Some epitopes may be fixation-sensitive; test various fixation conditions

  • Post-fixation permeabilization may be required for intracellular targets

• Blocking protocol optimization:

  • Zebrafish-specific blocking solutions (5-10% normal serum from the same species as secondary antibody)

  • Include 0.1-0.3% Triton X-100 for permeabilization if needed

  • Block for at least 1 hour at room temperature before antibody incubation

How does the conjugation process affect antibody stability and performance?

The FITC conjugation process can impact antibody characteristics in several ways:

• Stability considerations:

  • FITC conjugation slightly reduces antibody stability

  • Half-life of FITC-conjugated antibodies is typically shorter than unconjugated versions

  • Solution: Store at recommended temperatures; aliquot to avoid freeze-thaw cycles; add stabilizing proteins (BSA 0.1-1%)

• Impact on binding kinetics:

  • Increasing FITC labeling can affect binding affinity

  • Studies using isothermal titration calorimetry (ITC) have shown that extensive labeling can alter thermodynamic parameters of antibody-antigen binding

  • Heavily labeled antibodies may show decreased kon rates (association rates)

• Effect on protein conformation:

  • FITC conjugation to lysine residues near the antigen-binding site may interfere with recognition

  • Differential scanning fluorimetry studies have shown that labeling can affect protein thermal stability

  • Solution: When first conjugating an antibody, compare a range of FITC-to-antibody ratios (typically 40-80 µg FITC per mg of antibody)

• Species-specific considerations:

  • Rabbit polyclonal antibodies (like the crfb12 antibody) may have different optimal conjugation conditions than mouse monoclonals

  • Polyclonal preparations contain antibodies with variable FITC incorporation rates

  • Solution: Purify IgG fraction before conjugation; characterize batch-to-batch variation

How does crfb12 Antibody, FITC conjugated compare with other fluorophore conjugates for zebrafish research?

Different fluorophore conjugates offer distinct advantages in zebrafish research contexts:

FITC conjugates are particularly advantageous for:

• Well-established flow cytometry protocols

• Wide availability of compatible filter sets

• Lower cost compared to newer fluorophores

• Compatibility with most common flow cytometers and fluorescence microscopes

What are the latest methodological advances in FITC antibody conjugation relevant to zebrafish researchers?

Recent advancements have improved FITC conjugation efficiency and application:

• Rapid conjugation kits:

  • Lightning-Link® and similar technologies enable conjugation in under 20 minutes

  • Minimal hands-on time (approximately 30 seconds)

  • 100% antibody recovery, unlike traditional methods

  • Particularly useful for precious antibody samples

• Site-specific conjugation:

  • Rather than random lysine labeling, site-specific conjugation targets engineered sites

  • Preserves antigen-binding regions

  • Provides consistent fluorophore/protein ratios

  • Enables creation of homogeneous conjugates

• Optimization of fluorophore density:

  • Advanced characterization of optimal F/P ratios using differential scanning fluorimetry

  • Novel use of differential scanning fluorimetry without extrinsic fluorophores

  • Measurement of antigen binding via isothermal titration calorimetry (ITC)

• Enhanced conjugate purification:

  • Gradient DEAE Sephadex chromatography separates optimally labeled antibodies

  • Removes both under-labeled and over-labeled proteins

  • Improves signal-to-noise ratio in downstream applications

How can I integrate crfb12 Antibody, FITC conjugated into multiplex imaging platforms for comprehensive zebrafish immune studies?

Advanced multiplex platforms can maximize information from valuable zebrafish samples:

• Cyclic immunofluorescence approaches:

  • Sequential staining-imaging-bleaching cycles

  • Start with FITC conjugates (more easily bleached)

  • Follow with more photostable fluorophores

  • Can achieve 10+ parameters on the same tissue section

• Mass cytometry adaptation:

  • Convert FITC-based flow protocols to mass cytometry (CyTOF)

  • Use metal-tagged antibodies against the same targets

  • Enables 40+ parameter analysis without compensation issues

  • Particularly valuable for rare immune cell populations

• Spatial transcriptomics integration:

  • Combine FITC-based immunophenotyping with spatial transcriptomics

  • First identify crfb12+ cells by immunofluorescence

  • Then perform in situ sequencing or similar approaches

  • Creates multi-omic profiles with spatial context

• Microfluidic-based single-cell analysis:

  • Flow sorting of crfb12+ cells using FITC conjugate

  • Followed by droplet-based single-cell sequencing

  • Links protein expression with transcriptomic signatures

  • Reveals heterogeneity within seemingly homogeneous populations