VIPR2 Antibody, FITC conjugated

What is VIPR2 and what biological functions does it mediate?

VIPR2 (Vasoactive Intestinal Peptide Receptor 2) is a G protein-coupled receptor that belongs to the vasoactive intestinal peptide receptor family. It functions as a receptor for vasoactive intestinal peptide (VIP), a neuropeptide with diverse physiological roles. VIPR2 mediates multiple critical biological processes, including:

• Regulation of circadian rhythm

• Modulation of neurotransmission

• Immune response regulation

• Smooth muscle relaxation

• Exocrine and endocrine secretion regulation

• Water and ion homeostasis

Dysregulation of VIPR2 signaling has been implicated in various neurological and endocrine disorders, making it a promising target for therapeutic development. Understanding the function of VIPR2 is crucial for elucidating its role in these diseases and potentially identifying novel treatment strategies .

How does FITC conjugation affect the functionality of VIPR2 antibodies?

FITC (Fluorescein Isothiocyanate) conjugation provides direct fluorescent labeling of VIPR2 antibodies, enabling visualization without secondary detection reagents. This modification affects antibody functionality in several ways:

• Enables direct detection in flow cytometry, immunofluorescence, and live cell imaging

• Eliminates potential cross-reactivity issues from secondary antibodies

• May slightly reduce binding affinity compared to unconjugated antibodies due to steric hindrance

• Can affect the shelf-life and stability (FITC is sensitive to photobleaching)

• Optimal pH for FITC fluorescence is 8.0, which may influence experimental conditions

For applications requiring quantitative analysis, researchers should be aware that the degree of labeling (number of FITC molecules per antibody) can affect both binding capacity and signal intensity .

What are the validated applications for VIPR2 Antibody, FITC conjugated?

VIPR2 Antibody, FITC conjugated has been validated for several research applications, including:

The antibody shows high reactivity with human samples and has cross-reactivity with rat and mouse VIPR2, making it versatile for comparative studies across species. For optimal results, validation in your specific experimental system is recommended .

What sample types can be effectively analyzed with VIPR2 Antibody, FITC conjugated?

VIPR2 Antibody, FITC conjugated can be used effectively with various biological sample types:

• Cell lines expressing VIPR2 (particularly neuronal, endocrine, and immune cell lineages)

• Primary tissue sections (brain, pancreas, intestine, immune tissues)

• Peripheral blood mononuclear cells (PBMCs)

• Isolated T-cells (important for immune regulation studies)

• Tissue microarrays for high-throughput screening

When working with tissue samples, proper fixation protocols that preserve epitope accessibility while maintaining tissue architecture are critical. For cell suspensions, gentle fixation methods that don't disrupt membrane proteins are recommended .

What are the optimal sample preparation protocols for VIPR2 detection?

For optimal detection of VIPR2 using FITC-conjugated antibodies, sample preparation protocols should be tailored to the application:

For flow cytometry:

• Use single-cell suspensions (1-5×10^6 cells/mL)

• Gentle fixation with 0.5-2% paraformaldehyde

• Permeabilization only if detecting intracellular epitopes

• Blocking with 5% normal serum from the same species as secondary antibody

For immunofluorescence:

• 4% paraformaldehyde fixation (10-15 minutes)

• Antigen retrieval if using paraffin sections

• 0.1-0.3% Triton X-100 for permeabilization

• Extended blocking (1-2 hours) to reduce background

• Counterstaining nuclei with DAPI

For all applications, inclusion of proper negative controls (isotype control, FITC-conjugated non-specific antibody) is essential to establish specificity of staining patterns .

How can VIPR2 Antibody, FITC conjugated be used to investigate circadian rhythm mechanisms?

VIPR2 is critically involved in circadian rhythm regulation, particularly in the suprachiasmatic nucleus (SCN). FITC-conjugated VIPR2 antibodies enable precise investigation of this system through:

• Temporal expression profiling: Track VIPR2 expression throughout circadian cycles using timed tissue collection and flow cytometry quantification

• Co-localization studies: Combine with markers for other circadian regulators (PER, CRY, CLOCK) using multi-channel immunofluorescence

• Ex vivo SCN slice cultures: Visualize real-time VIPR2 dynamics in response to phase-shifting stimuli

• Receptor trafficking: Monitor internalization and recycling of VIPR2 following VIP stimulation

A methodological approach would involve:

• Harvest SCN tissue at 4-hour intervals across a 24-hour period

• Process for either flow cytometry or tissue sectioning

• Label with VIPR2 Antibody, FITC conjugated (1:100 dilution)

• Quantify fluorescence intensity and localization patterns

• Correlate with circadian time points and behavioral outputs

This approach has revealed that VIPR2 undergoes rhythmic expression changes with peak levels during the subjective night, coinciding with periods of maximal VIP sensitivity .

What experimental controls are critical when using VIPR2 Antibody, FITC conjugated in flow cytometry?

When using VIPR2 Antibody, FITC conjugated for flow cytometry, the following controls are essential for reliable data interpretation:

Additionally, titration experiments should be performed to determine optimal antibody concentration, as both insufficient and excessive antibody can compromise data quality. For multipanel flow cytometry, spectral overlap between FITC and other fluorophores (particularly PE) should be carefully compensated .

How can the specificity of VIPR2 Antibody, FITC conjugated be validated in neurological tissue samples?

Validating VIPR2 Antibody specificity in neurological tissues requires a multi-faceted approach:

• Comparative immunohistochemistry:

  • Parallel staining with multiple VIPR2 antibodies targeting different epitopes

  • Comparison of staining patterns with published atlas data

  • Cross-validation with in situ hybridization for VIPR2 mRNA

• Knockout/knockdown validation:

  • Use of VIPR2 knockout tissues as negative controls

  • RNAi-mediated knockdown in primary neuronal cultures

  • CRISPR-Cas9 edited cell lines or tissues

• Peptide competition assays:

  • Pre-incubation of antibody with excess immunizing peptide

  • Titration of blocking peptide to determine concentration-dependent inhibition

  • Use of related but distinct peptides to confirm epitope specificity

• Western blot correlation:

  • Confirmation that tissue lysates show bands of expected molecular weight

  • Correlation between band intensity and immunofluorescence signal strength

This comprehensive validation approach ensures that observed staining truly represents VIPR2 distribution, which is particularly critical in neurological tissues where non-specific binding can be problematic .

What is the relationship between VIPR2 expression and T-cell activation in anti-leukemic responses?

Recent research has revealed important connections between VIPR2 signaling and T-cell-mediated anti-leukemic activity:

VIPR2 signaling typically exerts immunosuppressive effects on T cells. When VIPR2 is engaged by VIP, it triggers cAMP production that dampens T-cell activation. Conversely, VIPR2 antagonism enhances T-cell responses against leukemia cells through multiple mechanisms:

• Increased T-cell proliferation following activation

• Enhanced production of pro-inflammatory cytokines (IFN-γ, TNF-α)

• Augmented cytotoxic activity against leukemia cells

• Reduced regulatory T-cell suppressive function

• Improved memory T-cell formation

Experimental evidence shows that VIP antagonists (VIPhyb) enhance T-cell activation and induce T-cell-dependent anti-leukemic activity in murine models of acute myeloid leukemia. The efficacy of these antagonists correlates positively with their predicted binding affinity to VIP receptors .

FITC-conjugated VIPR2 antibodies can be used to:

• Monitor receptor expression during different phases of T-cell activation

• Sort VIPR2-high and VIPR2-low T-cell populations for functional studies

• Track receptor internalization following antagonist treatment

• Correlate VIPR2 expression with anti-tumor activity in patient samples

These approaches have identified ANT308 and ANT195 as promising VIP-R antagonists with enhanced potency for inducing anti-leukemia immune responses .

How do research applications of VIPR2 antagonists differ from those using VIPR2 antibodies?

VIPR2 antagonists and VIPR2 antibodies represent complementary but distinct research tools with different applications and mechanisms:

The development of C-terminal sequence variations of VIPhyb has yielded improved VIP-R antagonists with enhanced receptor binding and plasma stability. In contrast, FITC-conjugated VIPR2 antibodies are primarily analytical tools that enable visualization and quantification of the receptor in research samples.

For comprehensive studies, researchers often use both approaches: antagonists to modulate function and antibodies to monitor expression and localization changes resulting from that modulation. This combination has proven particularly valuable in cancer immunology research investigating how VIPR2 signaling affects T-cell responses to malignancies .

What are the optimal storage and handling conditions for maintaining VIPR2 Antibody, FITC conjugated activity?

To maintain optimal activity of VIPR2 Antibody, FITC conjugated, adhere to these storage and handling guidelines:

• Store at -20°C in small aliquots to minimize freeze-thaw cycles

• Protect from light using amber tubes or by wrapping in aluminum foil

• Store in stabilizing buffer (typically pH 7.4 PBS with 0.05% NaN3 and 40% glycerol)

• When working with the antibody, keep on ice and protected from direct light

• Centrifuge briefly before opening vial to collect solution at the bottom

• Do not dilute until immediately before use

• Use within 12 months for optimal results

FITC conjugates are particularly sensitive to photobleaching, so minimize exposure to light during all handling steps. For long-term storage beyond 12 months, -80°C is recommended. Always verify activity with positive controls before use in critical experiments .

How can multiplexing with VIPR2 Antibody, FITC conjugated be optimized for co-localization studies?

Optimizing multiplexed immunofluorescence with FITC-conjugated VIPR2 antibody requires careful consideration of fluorophore combinations and staining protocols:

• Fluorophore selection strategies:

  • Pair FITC (excitation: 495nm, emission: 519nm) with fluorophores having minimal spectral overlap

  • Recommended partners: Cy5 (far-red), APC (red), or coumarin derivatives (blue)

  • Avoid PE or rhodamine derivatives which have significant overlap with FITC

• Sequential staining protocol:

  • Apply VIPR2 Antibody, FITC conjugated first (1:100 dilution)

  • Wash thoroughly (3-5 times with PBS + 0.05% Tween-20)

  • Block with 2% normal serum from host of second primary antibody

  • Apply second primary antibody

  • Use fluorophore-conjugated secondary antibody for detecting second primary

• Controls for co-localization studies:

  • Single-color controls for each fluorophore

  • Isotype controls for each primary antibody

  • Absorption controls (pre-incubation with blocking peptides)

  • Co-localization threshold controls using known interacting and non-interacting proteins

For optimal results, confocal microscopy with sequential scanning is preferred over wide-field fluorescence to minimize bleed-through. Post-acquisition processing should include background subtraction and careful thresholding based on control samples .

What troubleshooting approaches are recommended for weak or non-specific VIPR2 staining?

When encountering issues with VIPR2 Antibody, FITC conjugated staining, consider these systematic troubleshooting approaches:

For weak signal:

• Increase antibody concentration (try 2-fold increments)

• Extend incubation time (overnight at 4°C)

• Optimize fixation (try shorter fixation times to preserve epitopes)

• Enhance antigen retrieval (test multiple methods: citrate, EDTA, enzymatic)

• Use signal amplification systems (tyramide signal amplification)

• Check sample viability and VIPR2 expression level in your specific sample type

For high background/non-specific staining:

• Increase blocking duration and concentration (5-10% normal serum, 2 hours)

• Add 0.1-0.3% Triton X-100 to blocking solution

• Include 0.1-0.3% BSA in all antibody dilution buffers

• Increase wash steps (5-6 washes, 5 minutes each)

• Pre-adsorb antibody with tissue powder from non-expressing tissues

• Include human Fc block when working with human samples

For autofluorescence issues:

• Include Sudan Black B treatment (0.1% in 70% ethanol, 20 minutes)

• Use specialized autofluorescence quenching reagents

• Employ spectral unmixing during confocal microscopy acquisition

• Consider time-resolved fluorescence to separate FITC signal from autofluorescence

Documentation of all optimization steps in a laboratory notebook will facilitate reproducible protocols for future experiments .

How can VIPR2 Antibody, FITC conjugated contribute to neurological disorder research?

VIPR2 Antibody, FITC conjugated provides valuable tools for investigating neurological disorders through several methodological approaches:

• Receptor distribution mapping:

  • Comparative analysis of VIPR2 expression in healthy vs. diseased brain tissue

  • High-resolution mapping of receptor localization in specific neuronal populations

  • Quantitative assessment of receptor density changes during disease progression

• Schizophrenia research applications:

  • The VIPR2 gene (designated as SCZD16 in some databases) has been implicated in schizophrenia

  • Fluorescent antibodies enable visualization of altered receptor distribution in patient-derived samples

  • Flow cytometric quantification of VIPR2 levels in peripheral immune cells as potential biomarkers

• Circadian rhythm disorder investigations:

  • Visualization of altered VIPR2 expression in suprachiasmatic nucleus tissue

  • Correlation of receptor distribution with sleep-wake cycle abnormalities

  • Monitoring changes in receptor trafficking following chronobiotic treatments

• Neurodevelopmental studies:

  • Tracking VIPR2 expression changes during critical periods of brain development

  • Investigation of receptor clustering in developing neuronal networks

  • Analysis of VIPR2-associated signaling during neurite outgrowth and synaptogenesis

These applications rely on the specificity of FITC-conjugated VIPR2 antibodies for visualizing subtle changes in receptor distribution that may contribute to neurological dysfunction .

What methodological approaches can be used to study VIPR2 internalization and trafficking?

Studying VIPR2 internalization and trafficking using FITC-conjugated antibodies requires specialized techniques to capture receptor dynamics:

• Live-cell imaging approaches:

  • Pulse-chase labeling with VIPR2 Antibody, FITC conjugated

  • Time-lapse confocal microscopy to track receptor movement

  • Photobleaching techniques (FRAP/FLIP) to measure mobility

• Endocytic pathway characterization:

  • Co-localization with endosomal markers (Rab5, Rab7, Rab11)

  • Pharmacological inhibitors of different endocytic routes

  • Dynamin inhibitors to block clathrin-dependent internalization

• Quantitative internalization assays:

  • Acid wash removal of surface-bound antibody

  • Flow cytometry to measure internalization rate

  • Biotinylation-based biochemical assays

• Recycling vs. degradation fate determination:

  • Dual-pulse labeling with different color conjugates

  • Co-localization with lysosomal markers (LAMP1)

  • Inhibitors of lysosomal degradation (chloroquine, bafilomycin A)

A typical protocol would involve:

• Surface labeling with VIPR2 Antibody, FITC conjugated at 4°C (prevents internalization)

• Washing to remove unbound antibody

• Warming to 37°C to permit internalization

• Imaging at defined time intervals (0, 5, 15, 30, 60 min)

• Quantification of surface vs. intracellular fluorescence

These approaches can reveal how VIPR2 trafficking is regulated in response to VIP stimulation or antagonist treatment, providing insights into receptor desensitization mechanisms .

How can VIPR2 Antibody, FITC conjugated be used in immunotherapy development research?

VIPR2 Antibody, FITC conjugated plays a critical role in immunotherapy development research through several methodological approaches:

• Target validation and expression profiling:

  • Flow cytometric screening of patient samples to identify VIPR2-high populations

  • Correlation of VIPR2 expression with immunosuppressive tumor microenvironments

  • Isolation of VIPR2+ cells for functional characterization

• Therapeutic response monitoring:

  • Before/after treatment analysis of VIPR2 expression on immune cells

  • Real-time monitoring of receptor occupancy during VIP antagonist therapy

  • Correlation of receptor modulation with clinical outcomes

• Mechanism of action studies:

  • Visualization of VIPR2 distribution changes following antagonist treatment

  • Assessment of receptor clustering and signaling complex formation

  • Co-localization with downstream signaling molecules

• Companion diagnostic development:

  • Standardization of VIPR2 detection protocols for patient stratification

  • Threshold determination for predicting response to VIP-targeted therapies

  • Validation across different tissue types and disease states

Research has demonstrated that VIP antagonists enhance T-cell activation and induce T-cell-dependent anti-leukemic activity. FITC-conjugated VIPR2 antibodies enable monitoring of receptor expression and distribution changes during antagonist treatment, providing mechanistic insights that guide further therapeutic development .

What approaches can be used to correlate VIPR2 expression with functional outcomes in tissue samples?

Correlating VIPR2 expression with functional outcomes requires integrated methodological approaches:

• Sequential tissue analysis workflow:

  • Collect matched tissue samples for both staining and functional assays

  • Section tissue into adjacent slices for different analyses

  • Perform VIPR2 immunofluorescence with FITC-conjugated antibody

  • Conduct functional assays on matched sections (enzyme activity, electrophysiology, etc.)

  • Map expression patterns to functional readouts using image registration

• Single-cell analysis techniques:

  • Flow cytometric sorting of cells based on VIPR2-FITC signal intensity

  • Functional assessment of VIPR2-high vs. VIPR2-low populations

  • Correlation of receptor level with cellular responsiveness to VIP

  • Integration with single-cell transcriptomics for comprehensive profiling

• Ex vivo tissue culture approaches:

  • Precision-cut tissue slices maintained in culture

  • VIPR2 visualization using FITC-conjugated antibody

  • Functional testing with receptor agonists/antagonists

  • Time-lapse correlation of receptor dynamics with functional outputs

• In situ hybridization correlation:

  • RNAscope for VIPR2 mRNA detection on adjacent sections

  • Correlation of protein expression with transcript levels

  • Assessment of post-transcriptional regulation

These approaches have revealed that in leukemia research, T cells with higher VIPR2 expression show differential responses to VIP antagonist treatment, with expression levels correlating with anti-tumor activity potential .

What are the cutting-edge applications of VIPR2 Antibody, FITC conjugated in cancer immunology?

VIPR2 Antibody, FITC conjugated is enabling several cutting-edge applications in cancer immunology research:

• Immune checkpoint modulation studies:

  • VIPR2 signaling functions as an immune checkpoint in the tumor microenvironment

  • FITC-conjugated antibodies enable visualization of receptor distribution on tumor-infiltrating lymphocytes

  • Flow cytometric analysis of VIPR2 expression correlates with T-cell exhaustion markers

• CAR-T cell engineering applications:

  • Selection of VIPR2-low T cells for CAR modification (enhanced persistence)

  • Monitoring VIPR2 expression changes during CAR-T manufacturing

  • Correlation of pre-infusion VIPR2 levels with in vivo efficacy

• Combination therapy development:

  • Visualization of VIPR2 modulation when combining VIP antagonists with other immunotherapies

  • Assessment of receptor redistribution following checkpoint blockade

  • Identification of optimal sequencing for multi-modal immunotherapy

• Biomarker development:

  • VIPR2 expression profiling across cancer types to identify responsive subsets

  • Correlation of receptor levels with tumor microenvironment immune composition

  • Development of predictive algorithms based on VIPR2 distribution patterns

Recent research has demonstrated that VIP antagonists with improved docking scores for human VIP receptors VPAC1 and VPAC2 enhance T cell-dependent anti-leukemia responses. The predicted binding affinity of these antagonists correlates positively with their ability to augment T-cell proliferation and anti-leukemia activity, highlighting the importance of precisely characterizing VIPR2 expression and modulation in cancer immunotherapy development .

What future research directions are emerging for VIPR2 Antibody applications?

Emerging research directions for VIPR2 Antibody applications span multiple fields and methodological advances:

• Integration with advanced imaging technologies:

  • Super-resolution microscopy for nanoscale receptor clustering analysis

  • Expansion microscopy for improved spatial resolution of VIPR2 distribution

  • Light-sheet microscopy for whole-organ receptor mapping

  • Intravital imaging for real-time receptor dynamics in living tissues

• Multi-omics integration approaches:

  • Correlation of VIPR2 protein expression with spatial transcriptomics

  • Integrated proteomics to identify VIPR2 interaction networks

  • Metabolomics correlation with receptor signaling activity

• Novel therapeutic targeting strategies:

  • Development of VIPR2-targeted antibody-drug conjugates

  • Bispecific antibodies linking VIPR2 with immune effector cells

  • Nanoparticle-delivered VIP antagonists with improved pharmacokinetics

• Expanded disease applications:

  • Neurodegenerative disorders beyond circadian rhythm disruption

  • Inflammatory bowel disease and gut-brain axis investigation

  • Metabolic disorders with neuroendocrine components

• Advanced conjugation technologies:

  • Photoswitchable fluorophores for super-resolution applications

  • Quantum dot conjugation for improved stability and brightness

  • Multicolor VIPR2 antibody panels for multiplexed receptor family analysis