Avpr1b Antibody, HRP conjugated

What is Avpr1b and what biological systems is it primarily involved in?

Avpr1b (Arginine Vasopressin Receptor 1b) is a G-protein coupled receptor prominently expressed in hippocampal CA2 pyramidal cells and anterior pituitary corticotrophs. This receptor plays crucial roles in multiple biological systems, particularly in social behavior modulation, stress response, and hormonal regulation. Research has established Avpr1b as a key component in social recognition memory, social forms of aggression, and modulation of the hypothalamic-pituitary-adrenal (HPA) axis, especially under acute stress conditions . Additionally, recent studies have revealed Avpr1b's unexpected role in hematopoiesis, particularly in red blood cell production and differentiation .

What are the key specifications of the Avpr1b Antibody, HRP conjugated?

The Avpr1b Antibody, HRP conjugated (catalog number CSB-PA002469LB01RA) is a rabbit polyclonal antibody specifically targeting the Avpr1b protein. Its key specifications include:

This antibody's HRP conjugation eliminates the need for secondary antibody incubation in immunodetection techniques, streamlining experimental protocols .

How does Avpr1b function in the stress response pathway?

Avpr1b functions as a critical regulator of the hypothalamic-pituitary-adrenal (HPA) axis, particularly during acute stress responses. When vasopressin binds to Avpr1b receptors on anterior pituitary corticotrophs, it triggers intracellular signaling cascades that stimulate the release of adrenocorticotropic hormone (ACTH). Pharmacological studies using the Avpr1b antagonist SSR149415 consistently demonstrate decreased plasma ACTH levels in response to various stressors, confirming Avpr1b's crucial role in this pathway .

Beyond HPA-axis activation, Avpr1b signaling significantly influences behavioral stress phenotypes. Treatment with SSR149415 produces anxiolytic effects across multiple behavioral paradigms, including reduced defensive attacks, decreased immobility in forced swim tests, increased tolerance in punished drinking tasks, and more open arm entries in elevated plus maze tests. These effects are often more potent than those observed with traditional anxiolytics like diazepam, highlighting Avpr1b's potential as a therapeutic target for stress-related disorders .

What are the optimal protocols for utilizing Avpr1b Antibody, HRP conjugated in Western blot analysis?

For optimal Western blot analysis using Avpr1b Antibody, HRP conjugated, researchers should follow this methodological approach:

• Sample Preparation: Prepare tissue lysates (appropriate tissues include rat lung and mouse kidney based on validated reactivity) with complete protease inhibitors.

• Protein Separation: Separate proteins via SDS-PAGE using standard conditions.

• Transfer: Transfer proteins to a PVDF or nitrocellulose membrane using established transfer parameters for G-protein coupled receptors.

• Blocking: Block the membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary Antibody Incubation: Dilute the Avpr1b Antibody, HRP conjugated at 1:200 in blocking buffer and incubate overnight at 4°C .

• Washing: Wash the membrane 3-5 times with TBST.

• Detection: Since the antibody is HRP-conjugated, proceed directly to chemiluminescent detection without secondary antibody incubation.

• Validation Control: For specificity validation, perform a parallel experiment using the antibody pre-incubated with Vasopressin V1B Receptor/AVPR1B Blocking Peptide (such as BLP-VR011) at a ratio of 1 μg peptide per 1 μg antibody .

This protocol has been validated for detection of Avpr1b in rat and mouse tissues, as demonstrated in controlled experiments showing signal neutralization when the antibody is pre-adsorbed with the blocking peptide .

How should researchers optimize immunohistochemistry protocols when using Avpr1b antibodies?

For immunohistochemistry applications with Avpr1b antibodies, researchers should implement the following optimized protocol:

• Tissue Preparation: Prepare 4-μm sections from formalin-fixed paraffin-embedded tissue and place on pre-cleaned, charged microscope slides .

• Deparaffinization and Rehydration: Process slides through xylene and graded alcohols, including three changes of 100% alcohol for 3 minutes each at room temperature .

• Antigen Retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes at 95-98°C. This critical step unmasks epitopes potentially obscured during fixation.

• Peroxidase Blocking: Block endogenous peroxidase activity with 3% hydrogen peroxide for 10 minutes.

• Protein Blocking: Apply protein block solution for 10 minutes, then drain without washing.

• Primary Antibody Application: Apply diluted Avpr1b antibody (optimal dilution should be determined empirically, typically 1:100 to 1:200) and incubate for 45 minutes at room temperature .

• Washing: Wash thoroughly with buffer.

• Detection System: For non-HRP conjugated antibodies, apply appropriate detection system. For HRP-conjugated antibodies, proceed directly to chromogen development.

• Chromogen Development: Apply DAB chromogen and monitor for optimal development.

• Counterstaining and Mounting: Counterstain with hematoxylin, dehydrate through graded alcohols, clear in xylene, and mount.

For specificity validation, parallel sections should be processed with either isotype control antibodies or primary antibody pre-adsorbed with blocking peptide. Researchers should note that Avpr1b shows distinctive expression patterns in pituitary corticotrophs and hippocampal CA2 pyramidal cells .

How can Avpr1b antibodies be utilized to investigate the receptor's role in hematopoiesis and erythropoiesis?

Recent breakthrough research has revealed Avpr1b's unexpected role in hematopoiesis, particularly in the proliferation and differentiation of red blood cells. To investigate this novel function, researchers can employ Avpr1b antibodies in the following methodological approaches:

• Immunophenotyping of Hematopoietic Cells: Use Avpr1b antibodies in flow cytometry or immunofluorescence to quantify and characterize Avpr1b expression across different hematopoietic stem and progenitor cell (HSPC) populations. This approach has confirmed that all three AVP receptors are expressed in human HSPCs, with AVPR1B appearing particularly important in erythropoiesis .

• Western Blot Analysis of Differentiation Stages: Apply Avpr1b antibodies in Western blot analysis of cell lysates from various stages of erythroid differentiation to track temporal changes in receptor expression. Previous studies have shown that "AVPR1B and AVPR2 receptors appeared to decrease during terminal differentiation of erythroid cells" .

• Colony Formation Assays: Incorporate Avpr1b antibodies as blocking agents in methylcellulose colony assays to assess the functional requirement of Avpr1b in erythroid colony formation. Research has demonstrated that AVP and AVPR1B agonists increase burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) numbers under specific conditions .

• Calcium Signaling Studies: Combine Avpr1b antibody-based receptor identification with calcium flux assays to investigate signaling mechanisms. Studies have shown that "both AVP and d(Cha4)-AVP, a human-specific AVPR1B agonist, increase calcium in the cytoplasm of human CD34+ cells" .

• Phosphorylation Analysis: Use Avpr1b antibodies in immunoprecipitation followed by phospho-specific antibody detection to investigate downstream signaling events. AVP has been shown to increase phosphorylation of signal transducer and activator of transcription 5 (STAT5), similar to erythropoietin (EPO) .

These methodological approaches can help delineate the specific contributions of Avpr1b signaling to erythropoiesis and potentially identify new therapeutic targets for anemia, particularly in patients with central diabetes insipidus who often present with unexplained anemia .

What experimental designs can elucidate the intersection of Avpr1b signaling with neuropsychiatric disorders?

To investigate Avpr1b's role in neuropsychiatric disorders, researchers can implement the following experimental designs utilizing Avpr1b antibodies:

• Comparative Expression Analysis: Use immunohistochemistry with Avpr1b antibodies to compare receptor expression in post-mortem brain tissue from patients with various neuropsychiatric disorders versus controls. Focus on the hippocampal CA2 region, where Avpr1b is prominently expressed .

• Conditional Knockout Models: Combine Avpr1b antibody-based validation with genetic models to assess the behavioral and physiological consequences of region-specific Avpr1b deletion or overexpression. This approach can help distinguish between pituitary and hippocampal Avpr1b functions.

• Pharmacological Intervention Studies: Use Avpr1b antibodies to validate target engagement in studies utilizing Avpr1b antagonists like SSR149415, which has shown "more potent anxiolytic and antidepressant effects during both punished drinking tasks and elevated plus maze tests, as compared to diazepam" .

• Stress-Induced Behavioral Paradigms: Apply Avpr1b antibodies in immunohistochemistry to assess stress-induced changes in receptor expression or localization in animal models subjected to various stressors, correlating these changes with behavioral outcomes.

• Receptor-Specific Signaling Analysis: Employ co-immunoprecipitation with Avpr1b antibodies followed by proteomic analysis to identify interaction partners and signaling pathways specific to neuropsychiatric conditions.

These experimental approaches can help elucidate how Avpr1b signaling contributes to neuropsychiatric disorders and identify potential therapeutic opportunities through modulation of this receptor system .

How can researchers validate the specificity of Avpr1b antibody signal in their experiments?

Validating antibody specificity is crucial for ensuring reliable results. For Avpr1b antibodies, researchers should implement the following comprehensive validation strategy:

• Pre-adsorption Controls: Perform parallel experiments using the Avpr1b antibody pre-incubated with a specific blocking peptide (such as BLP-VR011). Signal elimination or substantial reduction confirms specificity. For optimal results, use a ratio of 1 μg peptide per 1 μg antibody .

• Knockout/Knockdown Validation: When available, use samples from Avpr1b knockout animals or cells with siRNA-mediated Avpr1b knockdown as negative controls. Western blot analysis of these samples should show absence of the specific band detected in wild-type samples.

• Multiple Antibody Verification: Employ multiple antibodies targeting different epitopes of Avpr1b and compare detection patterns. Concordant results across different antibodies increase confidence in specificity.

• Positive Control Selection: Include validated positive control samples with known Avpr1b expression. For the HRP-conjugated Avpr1b antibody, rat lung and mouse kidney lysates have been validated as appropriate positive controls .

• Peptide Competition Gradient: Perform a dose-dependent competition assay with increasing concentrations of blocking peptide to demonstrate gradual signal reduction, which provides stronger evidence of specificity than a single concentration test.

• Molecular Weight Verification: Ensure that the detected band corresponds to the expected molecular weight of Avpr1b (approximately 47 kDa), accounting for potential post-translational modifications.

• Cross-reactivity Assessment: Test the antibody on tissues from species with different sequence homology to evaluate potential cross-reactivity. The sequence used for generating BLP-VR011, for example, corresponds to amino acid residues 399-412 of rat AVPR1B (Accession P48974) .

This multi-faceted validation approach ensures robust specificity verification and enhances data reliability and reproducibility.

What are the common pitfalls in Avpr1b detection and how can they be addressed?

Researchers frequently encounter several challenges when detecting Avpr1b. Here are the common pitfalls and methodological solutions:

• High Background in Western Blots

  • Problem: Non-specific binding causing high background signal.

  • Solution: Increase blocking time/concentration (use 5% BSA instead of milk for phospho-specific detection), optimize antibody dilution (start with 1:200 for Avpr1b-HRP conjugated antibody), and include 0.1% Tween-20 in washing buffers .

• Inconsistent Immunohistochemistry Results

  • Problem: Variable staining intensity across samples.

  • Solution: Standardize fixation protocol (overfixation can mask epitopes), optimize antigen retrieval (citrate buffer pH 6.0, 20 minutes at 95-98°C), and incubate primary antibody for precisely 45 minutes at controlled room temperature .

• Signal Loss During Storage

  • Problem: Diminished antibody performance over time.

  • Solution: Store the HRP-conjugated antibody at -20°C or preferably -80°C, avoid repeated freeze-thaw cycles, and aliquot the reconstituted antibody to minimize freeze-thaw events .

• Low Signal in Expected Positive Samples

  • Problem: Insufficient signal despite appropriate sample selection.

  • Solution: Verify protein loading amount (increase if necessary), test different antigen retrieval methods, and consider the diluent buffer composition (the antibody contains 50% Glycerol, 0.01M PBS, pH 7.4) .

• Cross-Reactivity Issues

  • Problem: Antibody detecting proteins other than Avpr1b.

  • Solution: Perform parallel detection with pre-adsorbed antibody, verify with species-appropriate positive controls, and check sequence homology for cross-species applications (the BLP-VR011 sequence shows 100% identity across human, gorilla, gibbon, and monkey, with 88% identity in marmoset, elephant, bat, and pig) .

• Receptor Conformational Changes

  • Problem: Sample preparation altering epitope accessibility.

  • Solution: Use non-denaturing conditions when possible, avoid harsh detergents, and ensure sample buffers maintain physiological pH to preserve receptor conformation.

By anticipating these common pitfalls and implementing the suggested methodological solutions, researchers can significantly enhance the reliability and reproducibility of their Avpr1b detection experiments.

How might Avpr1b antibodies contribute to developing novel therapeutic approaches for stress-related disorders?

Avpr1b antibodies can drive therapeutic innovation for stress-related disorders through several methodological approaches:

• Target Validation in Preclinical Models: Researchers can use Avpr1b antibodies in immunohistochemistry and Western blot analyses to validate Avpr1b expression in anxiety and depression models before and after treatment with potential therapeutic compounds. Studies have already demonstrated that antagonism of Avpr1b with SSR149415 produces potent anxiolytic and antidepressant effects in multiple behavioral paradigms, often exceeding the efficacy of traditional anxiolytics like diazepam .

• Receptor Occupancy Assays: Develop competitive binding assays with labeled Avpr1b antibodies to measure receptor occupancy by therapeutic candidates, providing critical pharmacodynamic data for dose optimization in preclinical and clinical studies.

• Biomarker Development: Apply Avpr1b antibodies in immunoassays to quantify soluble receptor fragments or associated proteins in biological fluids, potentially identifying stress-responsive biomarkers that could track disease progression or treatment response.

• Blood-Brain Barrier Penetration Studies: Use Avpr1b antibodies to assess central target engagement of peripherally administered Avpr1b modulators, addressing a key challenge in CNS drug development.

• Cell-Type Specific Drug Delivery: Conjugate therapeutic payloads to modified Avpr1b antibodies for targeted delivery to Avpr1b-expressing cells, potentially reducing side effects while maintaining efficacy.

• Allosteric Modulator Screening: Apply Avpr1b antibodies in conformational assays to identify compounds that modulate receptor function through allosteric mechanisms, potentially offering improved selectivity over orthosteric approaches.

What experimental approaches can elucidate the mechanistic relationship between Avpr1b signaling and hematopoiesis?

Recent discoveries linking Avpr1b to erythropoiesis open exciting research directions. Researchers can employ these experimental approaches to mechanistically characterize this relationship:

• Conditional Tissue-Specific Knockout Models: Generate hematopoietic-specific Avpr1b knockout models and use Avpr1b antibodies to confirm deletion efficacy. These models can then be challenged with anemia-inducing conditions to assess the specific contribution of Avpr1b to stress erythropoiesis.

• Signaling Pathway Dissection: Use phospho-specific antibodies in combination with Avpr1b antibodies to map the temporal sequence of signaling events following receptor activation. Research has shown that "AVP increases phosphorylation of signal transducer and activator of transcription 5, as erythropoietin (EPO) does," suggesting potential pathway convergence or cross-talk .

• Receptor Trafficking Analysis: Apply immunofluorescence with Avpr1b antibodies to track receptor internalization and recycling in hematopoietic progenitors following ligand binding, which may reveal unique aspects of signaling duration and intensity.

• Calcium Signaling Quantification: Combine calcium imaging with immunocytochemical identification of Avpr1b-positive cells to correlate receptor expression levels with calcium response magnitude. Studies have demonstrated that "both AVP and d(Cha4)-AVP, a human-specific AVPR1B agonist, increase calcium in the cytoplasm of human CD34+ cells" .

• Single-Cell Transcriptomics: Implement scRNA-seq on FACS-sorted Avpr1b-positive hematopoietic populations to identify unique transcriptional programs activated by Avpr1b signaling during erythroid differentiation.

• Ex Vivo Differentiation Assays: Culture hematopoietic stem cells with Avpr1b agonists, antagonists, or blocking antibodies while monitoring differentiation markers to establish direct causality between receptor signaling and erythroid commitment. Previous research found that "when AVP and the AVPR1B agonist d(Cha4)-AVP were added to the medium, they increased burst-forming unit-erythroid (BFU-E) but not colony-forming unit-erythroid (CFU-E) numbers" .

• In Vivo Recovery Models: Use Avpr1b antibodies to monitor receptor expression changes during recovery from sublethal irradiation or chemically-induced anemia, correlating with hematological parameters. Studies with AVP-deficient Brattleboro rats showed "delayed recovery of RBC numbers compared to control rats" after sublethal irradiation .

These methodological approaches will help elucidate the precise mechanisms by which Avpr1b signaling influences hematopoiesis, potentially leading to novel therapeutic strategies for anemia, particularly in patients with central diabetes insipidus who often present with unexplained anemia.