Avpr1b Antibody, FITC conjugated

What is AVPR1B and what are its alternative nomenclatures?

AVPR1B (Arginine Vasopressin Receptor 1B) is a receptor for arginine vasopressin whose activity is mediated by G proteins that activate a phosphatidyl-inositol-calcium second messenger system. The protein is also known by several alternative names including AVPR3, VPR3, Vasopressin V1b receptor, V1bR, AVPR V1b, AVPR V3, Antidiuretic hormone receptor 1b, and Vasopressin V3 receptor . AVPR1B is involved in crucial physiological processes and during SARS-CoV-2 infection may recognize and internalize the complex formed by AVP/Arg-vasopressin, SARS-CoV-2 spike protein, and secreted ACE2 through DNM2/dynamin 2-dependent endocytosis .

What are the primary applications for AVPR1B antibodies in research?

AVPR1B antibodies are employed in multiple research applications, with the most common being immunohistochemistry (IHC-P), Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and various immunofluorescence techniques (IF) . These antibodies are critical for studying AVPR1B expression patterns, localization, and function in different tissues. FITC-conjugated versions specifically enable direct fluorescence detection without requiring secondary antibodies, making them particularly valuable for multicolor immunofluorescence studies, flow cytometry, and confocal microscopy applications .

What tissue types have been validated for AVPR1B antibody detection?

AVPR1B antibodies have been validated for detection in several human tissues. These include pituitary anterior tissue (particularly important as AVPR1B plays a significant role in the pituitary), pancreatic tissue, and kidney tissue . For optimal results in immunohistochemistry applications, antigen retrieval with TE buffer (pH 9.0) is recommended, although citrate buffer (pH 6.0) may be used as an alternative .

What are the recommended storage conditions for FITC-conjugated AVPR1B antibodies?

FITC-conjugated antibodies, including AVPR1B antibodies, should generally be stored at -20°C for long-term storage, with protection from light to prevent photobleaching of the FITC fluorophore. Most preparations contain stabilizers like 50% glycerol and sometimes BSA (1%) . When working with the antibody, aliquoting is recommended to avoid repeated freeze/thaw cycles which can degrade both the antibody and the fluorophore conjugate. For short-term storage (up to one month), the antibody can be kept at 4°C, but must still be protected from light exposure .

What optimization steps are recommended when using FITC-conjugated AVPR1B antibody for immunofluorescence?

When optimizing FITC-conjugated AVPR1B antibody for immunofluorescence applications, researchers should consider the following methodological approach:

• Concentration optimization: Begin with a titration experiment testing dilutions ranging from 1:50 to 1:500 to determine optimal signal-to-noise ratio .

• Fixation method evaluation: Compare paraformaldehyde fixation (4%) with methanol fixation to determine which best preserves both the epitope and FITC fluorescence.

• Background reduction: Implement blocking with 5-10% normal serum from the same species as the secondary antibody used in other channels, plus 0.1-0.3% Triton X-100 for permeabilization.

• Photobleaching prevention: Use anti-fade mounting medium containing DAPI for nuclear counterstaining.

• Controls: Always include a negative control (no primary antibody) and if possible, a blocking peptide control to verify specificity .

Each of these parameters should be systematically tested and optimized for specific tissue types and experimental designs.

How can researchers troubleshoot weak or absent signals when using FITC-conjugated AVPR1B antibodies?

When encountering weak or absent signals with FITC-conjugated AVPR1B antibodies, a systematic troubleshooting approach should be followed:

Additionally, researchers should verify target protein expression in their specific sample type and consider using positive control tissues like human anterior pituitary, pancreas, or kidney tissue where AVPR1B expression has been confirmed .

What are the critical considerations for multiplexing FITC-conjugated AVPR1B antibody with other fluorophore-conjugated antibodies?

When designing multiplex immunofluorescence experiments incorporating FITC-conjugated AVPR1B antibody, researchers should consider:

• Spectral overlap: FITC (excitation ~495nm, emission ~520nm) has potential spectral overlap with other green fluorophores. Design panels using fluorophores with well-separated emission spectra (e.g., FITC, TRITC/Cy3, Cy5).

• Antibody compatibility: Ensure all primary antibodies in the panel are raised in different host species or use different isotypes if from the same species.

• Sequential staining: For challenging combinations, implement sequential staining protocols with blocking steps between antibody applications.

• Cross-reactivity testing: Validate each antibody individually before combining to ensure specific binding.

• Signal balancing: Adjust antibody concentrations to achieve balanced signal intensities across all channels.

• Compensation controls: Include single-stained samples for each fluorophore to allow for spectral compensation during analysis .

Careful experimental design and rigorous controls are essential for generating reliable multiplexed immunofluorescence data.

How can FITC-conjugated AVPR1B antibodies be used to investigate the role of AVPR1B in SARS-CoV-2 infection?

Recent research has identified that during SARS-CoV-2 infection, AVPR1B may recognize and internalize a complex formed by AVP/Arg-vasopressin, SARS-CoV-2 spike protein, and secreted ACE2 through DNM2/dynamin 2-dependent endocytosis . FITC-conjugated AVPR1B antibodies provide powerful tools for investigating this phenomenon through:

• Co-localization studies: Using confocal microscopy to visualize AVPR1B (FITC-labeled) with spike protein and ACE2 (labeled with spectrally distinct fluorophores).

• Internalization kinetics: Time-course experiments tracking the movement of AVPR1B from membrane to intracellular locations following viral exposure.

• Receptor trafficking: Live-cell imaging to monitor real-time receptor dynamics during viral entry.

• Inhibitor screening: Testing compounds that may disrupt the AVPR1B-mediated viral entry pathway.

These approaches could help elucidate the mechanistic details of this novel viral entry pathway and potentially identify new therapeutic targets for COVID-19 treatment.

What experimental approaches can be used to validate AVPR1B antibody specificity in FITC-conjugated formats?

Validating the specificity of FITC-conjugated AVPR1B antibodies is crucial for generating reliable research data. Recommended validation approaches include:

• Peptide competition assays: Pre-incubate the antibody with excess immunizing peptide before application to samples; specific signals should be dramatically reduced.

• Knockout/knockdown controls: Test the antibody in samples where AVPR1B has been knocked out (CRISPR/Cas9) or knocked down (siRNA); specific signals should be absent or significantly reduced.

• Overexpression systems: Compare signals in cells with baseline expression versus those overexpressing AVPR1B; signal intensity should correlate with expression level.

• Western blot correlation: Confirm that IF/IHC patterns correlate with specific bands of expected molecular weight (46-47 kDa) in Western blot analyses .

• Multiple antibody verification: Compare staining patterns using antibodies targeting different epitopes of AVPR1B.

• Cross-reactivity testing: Evaluate signals in tissues known to express related receptors (e.g., AVPR1A, AVPR2) to ensure specificity.

These validation steps ensure that observed signals genuinely represent AVPR1B localization and expression.

How can researchers accurately quantify AVPR1B expression levels using FITC-conjugated antibodies in different experimental systems?

Accurate quantification of AVPR1B expression using FITC-conjugated antibodies requires rigorous methodology:

For all approaches, researchers should:

• Include calibration standards with known fluorophore quantities

• Implement background subtraction methods

• Account for potential autofluorescence (especially relevant for FITC's emission spectrum)

• Use consistent exposure/gain settings across all experimental groups

• Apply appropriate statistical analyses for comparisons

Standardization is critical - all samples should be processed simultaneously using identical protocols to minimize technical variability.

What controls are essential when using FITC-conjugated AVPR1B antibodies for immunofluorescence studies?

When designing immunofluorescence experiments with FITC-conjugated AVPR1B antibodies, the following controls are essential:

• Negative controls:

  • Isotype control: A FITC-conjugated non-specific antibody of the same isotype and concentration

  • Secondary-only control: Omitting primary antibody (less relevant for direct conjugates but still important)

  • Blocking peptide control: Primary antibody pre-incubated with excess immunizing peptide

• Positive controls:

  • Known positive tissue: Human anterior pituitary, pancreas, or kidney tissue

  • Overexpression system: Cells transfected to express AVPR1B

• Technical controls:

  • Autofluorescence control: Unstained sample to assess natural tissue fluorescence

  • Fixation control: Comparison of different fixation methods on signal intensity

  • Single-color controls: For spectral unmixing in multiplex experiments

These controls help distinguish specific from non-specific signals and provide confidence in experimental results.

How should researchers select between direct FITC-conjugated AVPR1B antibodies versus unconjugated primary with FITC-conjugated secondary antibodies?

The decision between using direct FITC-conjugated AVPR1B antibodies versus an unconjugated primary antibody with FITC-conjugated secondary depends on several experimental factors:

Direct FITC-conjugated AVPR1B advantages:

• Simplified protocol with fewer steps and shorter time requirements

• Reduced background from secondary antibody cross-reactivity

• Enabling of multiple primary antibodies from the same species in multiplex experiments

• More precise localization due to direct labeling

Unconjugated primary with FITC-secondary advantages:

• Signal amplification (multiple secondary antibodies can bind each primary)

• Greater flexibility to change fluorophores without purchasing new primary antibodies

• Potentially better retention of antibody affinity (conjugation can sometimes affect binding)

• Often more cost-effective for large-scale studies

Researchers should consider target abundance (low-expression targets may benefit from signal amplification with secondary antibodies) and experimental complexity (multiplex designs may require direct conjugates to avoid cross-reactivity) .

What are the unique considerations when using FITC-conjugated AVPR1B antibodies in live-cell imaging applications?

Live-cell imaging with FITC-conjugated AVPR1B antibodies presents distinct challenges and requires special considerations:

• Cell membrane permeability: Since AVPR1B is a membrane receptor with extracellular domains, antibodies targeting these regions can bind without permeabilization, while those targeting intracellular domains cannot access their epitopes in live cells.

• Photobleaching mitigation:

  • Use low-intensity illumination and shortest possible exposure times

  • Implement oxygen-scavenging systems in imaging media

  • Consider using pulse-chase approaches rather than continuous imaging

• Physiological conditions:

  • Maintain cells at 37°C and appropriate CO₂ levels

  • Use phenol red-free media to reduce background fluorescence

  • Buffer systems must maintain physiological pH without disrupting normal AVPR1B function

• Antibody internalization:

  • Account for potential antibody-induced receptor internalization

  • Consider potential functional effects of antibody binding on receptor signaling

  • Use Fab fragments rather than complete IgG when receptor crosslinking is a concern

• Temporal considerations: FITC photobleaches relatively quickly compared to other fluorophores, potentially limiting long-term imaging experiments.

These specialized approaches enable researchers to study dynamic AVPR1B processes including trafficking, internalization, and response to ligands or drugs in living systems.

How can researchers differentiate between specific AVPR1B signal and autofluorescence when using FITC-conjugated antibodies?

Distinguishing specific AVPR1B signal from autofluorescence when using FITC-conjugated antibodies requires multiple analytical approaches:

• Spectral analysis: FITC has a characteristic excitation/emission spectrum (495/520nm). True FITC signal should match this precisely, while autofluorescence often has broader spectral profiles. Spectral unmixing algorithms can help separate these signals.

• Negative control comparison: Systematically comparing stained samples with appropriate negative controls (unstained, isotype controls, blocking peptide controls) can identify autofluorescence.

• Multiple channel verification: True AVPR1B signal should be absent in other fluorescence channels (e.g., red, far-red), while autofluorescence often appears across multiple channels.

• Morphological assessment: AVPR1B shows characteristic subcellular localization (primarily membrane-associated with some cytoplasmic presence). Signals that don't match this expected pattern may represent autofluorescence .

• Quenching techniques: Chemical treatments like Sudan Black B or CuSO₄ can reduce autofluorescence without significantly affecting specific antibody signals.

Combining these approaches provides greater confidence in distinguishing true AVPR1B signal from autofluorescence.

What are the implications of AVPR1B alternative splicing variants for antibody detection and experimental design?

AVPR1B is subject to alternative splicing, producing protein isoforms that may affect antibody detection and experimental interpretation:

• Epitope availability: Different AVPR1B antibodies target distinct epitopes that may be present or absent in specific splice variants. When selecting FITC-conjugated AVPR1B antibodies, researchers should verify which domain is targeted and whether that region is conserved across relevant splice variants.

• Variant-specific detection strategies:

  • For comprehensive detection of all variants, target highly conserved domains

  • For variant-specific detection, target unique regions

  • Consider using multiple antibodies targeting different domains to distinguish variant expression

• Functional implications: Different splice variants may have distinct signaling properties, subcellular localizations, or protein interaction profiles. When studying AVPR1B function, researchers must consider which variants are being detected .

• Data interpretation considerations: Discrepancies between studies may result from antibodies detecting different AVPR1B variants. Researchers should explicitly state which antibody (and therefore which potential variants) were detected in their experiments.

Understanding the relationship between antibody epitopes and AVPR1B splice variants is crucial for accurate experimental design and interpretation.

How should researchers approach contradictory results obtained with different AVPR1B antibodies?

When researchers encounter contradictory results using different AVPR1B antibodies (including FITC-conjugated versions), a systematic troubleshooting approach is necessary:

• Epitope mapping comparison:

  • Determine which domains of AVPR1B each antibody targets

  • Assess whether epitopes might be differentially accessible in various experimental conditions

  • Consider whether post-translational modifications might affect epitope recognition

• Validation status review:

  • Evaluate the validation data for each antibody

  • Consider whether antibodies have been validated in the specific application and tissue being studied

  • Review literature for reported cross-reactivity or specificity issues

• Experimental conditions analysis:

  • Compare fixation methods, which can dramatically affect epitope accessibility

  • Review antigen retrieval approaches (TE buffer pH 9.0 versus citrate buffer pH 6.0)

  • Consider buffer composition differences that might affect antibody binding

• Resolution strategies:

  • Implement knockout/knockdown controls with each antibody

  • Use orthogonal detection methods (e.g., mRNA analysis, mass spectrometry)

  • Consider probing for AVPR1B interaction partners as indirect validation

  • Report discrepancies transparently in publications to advance field knowledge

Contradictory results often reflect biological complexity rather than technical failure and may lead to important discoveries about protein regulation or structure.