HRH4 Antibody, FITC conjugated

What is the HRH4 receptor and why is it significant in immunological research?

HRH4 (Histamine H4 receptor) belongs to the family 1 of G protein-coupled receptors (GPCRs) and has the highest homology to histamine receptor H3 among known G protein-coupled receptors. The receptor mediates histamine signaling in peripheral tissues and displays significant constitutive activity even in the absence of agonist stimulation . HRH4's importance in immunological research stems from its restricted expression pattern primarily in hematopoietic cells and its role in mediating various immunomodulatory effects. The receptor has been reported in human blood peripheral leukocytes, bone marrow, colon, liver, lung, small intestine, spleen, testis, thymus, tonsil, and trachea . This distribution pattern makes it a critical target for studying allergic and inflammatory conditions, particularly because it influences T cell function, cytokine production, and immune cell chemotaxis .

What are the key characteristics of FITC-conjugated HRH4 antibodies?

FITC-conjugated HRH4 antibodies are polyclonal antibodies typically raised in rabbits against human HRH4 receptor protein or peptide sequences. The conjugation with FITC (Fluorescein Isothiocyanate) provides a fluorescent tag that enables direct visualization in applications like immunofluorescence and flow cytometry without requiring secondary antibodies. These antibodies specifically recognize human HRH4 receptors and are supplied in liquid form in buffers containing preservatives like 0.03% Proclin 300 and stabilizers such as 50% glycerol in PBS (pH 7.4) . They are typically purified using Protein G affinity chromatography with >95% purity and require storage at -20°C or -80°C to maintain stability and avoid repeated freeze-thaw cycles . The immunogen for these antibodies is often recombinant Human Histamine H4 receptor protein, particularly regions such as amino acids 204-292 or synthetic peptides derived from human HRH4 .

How should researchers optimize sample preparation for HRH4 antibody immunofluorescence staining?

For optimal immunofluorescence staining with FITC-conjugated HRH4 antibodies, researchers should implement a systematic approach to sample preparation. Begin with proper fixation—4% paraformaldehyde for cells or formalin-fixed paraffin-embedded tissues—ensuring preservation of antigen structure while maintaining cellular morphology. For FFPE tissues, complete antigen retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 8.0) with heat treatment to expose epitopes potentially masked during fixation .

Blocking is critical to reduce non-specific binding, particularly given concerns about commercial HRH4 antibody specificity. Use 5-10% normal serum from a species unrelated to both the antibody host and target tissue, combined with 0.1-0.3% Triton X-100 for membrane permeabilization in a 1-2 hour incubation at room temperature . When applying the FITC-conjugated HRH4 antibody, the recommended dilution range is typically 1:50-200 for immunofluorescence applications . Incubate sections overnight at 4°C in a humidified chamber protected from light to prevent photobleaching of the FITC fluorophore.

Include appropriate controls in each experiment: negative controls (omitting primary antibody), isotype controls (using non-specific rabbit IgG-FITC), and positive controls using tissues known to express HRH4 (such as human lymph nodes) . These methodological considerations will help minimize potential nonspecific binding effects that have been reported with commercial HRH4 antibodies .

How can researchers reliably differentiate between HRH4 expression in regulatory T cells versus conventional CD4+ T cells?

Distinguishing HRH4 expression between regulatory T cells (Tregs) and conventional CD4+ T cells requires a multi-faceted approach due to the differential expression patterns observed in these cell populations. Research has demonstrated that HRH4 mRNA levels are significantly higher in CD4+CD25+Foxp3+ Tregs compared to conventional CD4+CD25-Foxp3- T cells . To reliably quantify this difference, researchers should implement a three-pronged strategy:

First, employ quantitative RT-PCR to assess HRH4 mRNA expression using properly validated primers specific to human HRH4, with normalization to stable endogenous control genes. This approach allows for precise quantification of transcript levels between isolated cell populations. Second, implement multi-parameter flow cytometry using FITC-conjugated HRH4 antibodies in combination with fluorescently-labeled antibodies against CD4, CD25, and intracellular Foxp3 to identify distinct T cell subsets. This permits simultaneous assessment of surface HRH4 protein expression and regulatory T cell markers at the single-cell level .

Third, validate these findings using functional assays that assess HRH4 signaling capacity through calcium mobilization assays or cAMP inhibition in response to selective H4R agonists like 4-methyl histamine. When implementing this protocol, researchers must carefully isolate pure cell populations using magnetic separation or flow cytometry-based sorting to avoid contamination between regulatory and conventional T cell populations, which could confound expression analysis results .

What experimental approaches can resolve the conflicting data regarding HRH4's role in inflammatory responses?

Resolving the conflicting data regarding HRH4's role in inflammatory responses requires comprehensive experimental strategies that address the current paradoxical findings where both pro-inflammatory and anti-inflammatory functions have been reported . A systematic approach should include:

A comprehensive cross-species analysis comparing HRH4 function in different model organisms and human samples, as species-specific differences may account for some conflicting results. This should involve parallel studies using both genetic approaches (HRH4 knockout models) and pharmacological interventions (selective antagonists like JNJ 7777120 and agonists like 4-methyl histamine) within the same disease models . Time-course studies are essential to determine whether HRH4 exhibits different functions at various stages of inflammation, potentially explaining contradictory observations where timing of intervention varied between studies.

Researchers should implement tissue-specific conditional knockout models to delineate the cell type-specific contributions of HRH4 to inflammatory responses. This approach would help determine whether HRH4 expression on different immune cell populations (mast cells, Tregs, eosinophils) mediates distinct and potentially opposing effects . Additionally, investigating the signaling pathway specificity is critical—researchers should examine whether HRH4 coupling to different downstream effectors (G-protein dependent versus β-arrestin pathways) under different conditions might explain the divergent functional outcomes .

Finally, conducting detailed single-cell transcriptomic analyses on various immune cell populations in inflammatory conditions will provide insight into whether HRH4 expression and function varies across distinct cellular subtypes and disease contexts .

How can researchers effectively validate HRH4 antibody specificity for accurate immunophenotyping studies?

Validating HRH4 antibody specificity is crucial for immunophenotyping studies, especially considering documented concerns about commercial antibody reliability . A comprehensive validation strategy must include multiple complementary approaches:

First, perform genetic validation using HRH4 knockout models or CRISPR/Cas9-mediated HRH4 deletion in human cell lines. Compare antibody staining patterns between wild-type and HRH4-deficient samples using the same experimental conditions. True specificity is confirmed when staining is absent in knockout samples . Second, employ peptide competition assays where the immunizing peptide used to generate the antibody is pre-incubated with the FITC-conjugated HRH4 antibody before sample application. Specific antibody binding should be significantly reduced or eliminated when the immunizing peptide blocks the antibody's antigen-binding sites .

Third, use orthogonal validation by correlating protein detection with mRNA expression. Implement in situ hybridization techniques alongside immunofluorescence to compare HRH4 transcript localization with protein detection patterns . Fourth, conduct Western blot analysis to confirm that the antibody detects a protein of the expected molecular weight (approximately 44 kDa for human HRH4), with additional validation using recombinant HRH4 protein as a positive control .

Finally, researchers should perform cross-platform validation by comparing results from multiple anti-HRH4 antibodies raised against different epitopes of the receptor. Consistent staining patterns across different antibodies provide strong evidence for specificity, while discrepancies suggest potential non-specific binding .

What are the optimal conditions for using FITC-conjugated HRH4 antibodies in flow cytometry applications?

For optimal flow cytometry performance using FITC-conjugated HRH4 antibodies, researchers should implement a carefully optimized protocol addressing several critical parameters. Begin with proper sample preparation by isolating fresh cells and maintaining viability above 90% (confirmed with viability dyes). Since HRH4 expression occurs primarily in hematopoietic cells, peripheral blood mononuclear cells or isolated leukocyte populations represent ideal targets .

For surface staining, use 1×10^6 cells per sample and implement a titratable antibody concentration, typically starting with dilutions between 1:50-1:200 to determine optimal signal-to-noise ratio . Include a pre-incubation step with Fc receptor blocking reagent (10-15 minutes at 4°C) to prevent non-specific binding, particularly important when examining myeloid cells with high Fc receptor expression . Perform staining in cell-friendly buffer (PBS with 1-2% BSA and 0.1% sodium azide) for 30 minutes at 4°C protected from light to preserve FITC fluorescence.

When designing multi-parameter panels, consider FITC's emission spectrum (peak ~520nm) to avoid spectral overlap with other fluorophores, particularly PE. When examining cells with low HRH4 expression, implement signal amplification strategies or consider alternative conjugates with higher quantum yield than FITC. For internal control validation, include fluorescence-minus-one (FMO) controls and isotype controls (rabbit IgG-FITC) to accurately set gates and account for background autofluorescence .

For intracellular HRH4 detection, implement fixation and permeabilization using commercial kits compatible with GPCRs, recognizing that overly harsh permeabilization may disrupt the native conformation of the receptor's epitopes, potentially affecting antibody recognition .

What methodological considerations are crucial when investigating HRH4-mediated signaling pathways in immune cells?

Investigating HRH4-mediated signaling pathways in immune cells requires specialized methodological considerations due to the receptor's complex coupling to multiple downstream effectors and its constitutive activity . Researchers should first establish appropriate cellular models, preferably primary human immune cells that naturally express HRH4 (such as mast cells, eosinophils, or T cells) rather than overexpression systems that may not recapitulate physiological signaling dynamics.

For examining G protein-dependent pathways, implement cAMP inhibition assays since HRH4 couples to Gi/o proteins. First stimulate cells with forskolin to elevate cAMP levels, then measure the inhibitory effect of HRH4-selective agonists using ELISA or FRET-based reporters . To confirm Gi/o protein involvement, pretreat cells with pertussis toxin (100-200 ng/ml, 16-18 hours), which should abolish HRH4-mediated cAMP inhibition. For calcium mobilization studies, load cells with fluorescent calcium indicators (Fluo-4/AM) and measure real-time changes in intracellular calcium following HRH4 stimulation using plate readers or flow cytometry .

To investigate β-arrestin recruitment, implement bioluminescence resonance energy transfer (BRET) or enzyme complementation assays that can detect protein-protein interactions between HRH4 and β-arrestin upon receptor activation . For downstream signaling pathways (MAPK/ERK, JAK-STAT, PI3K), use phospho-specific antibodies to detect activation-specific post-translational modifications by Western blotting or flow cytometry at multiple time points (5-60 minutes) to capture both rapid and delayed signaling events.

When using pharmacological tools, carefully select specific HRH4 agonists (4-methyl histamine) and antagonists (JNJ 7777120) at appropriate concentrations, recognizing that some compounds may have off-target effects at higher concentrations. Control experiments should include competitive antagonist studies to confirm signal specificity and siRNA-mediated HRH4 knockdown to validate antibody-detected signals .

How should researchers design experiments to investigate HRH4's role in regulatory T cell recruitment and function in inflammatory disease models?

Designing robust experiments to investigate HRH4's role in regulatory T cell recruitment and function in inflammatory disease models requires systematic approaches that address both mechanistic aspects and physiological relevance. Begin with careful selection of disease models—for allergic airway inflammation, both acute (ovalbumin-induced) and chronic (house dust mite-induced) murine models are appropriate, while for autoimmune conditions, experimental autoimmune encephalomyelitis models may be more suitable based on reported HRH4 involvement .

Implement both genetic and pharmacological approaches in parallel: utilize HRH4 knockout mice alongside conditional knockout models specific to Foxp3+ cells (Foxp3-Cre × HRH4-floxed) to distinguish between global HRH4 deficiency effects and Treg-specific HRH4 functions. Complement these with pharmacological studies using selective HRH4 agonists (4-methyl histamine) and antagonists (JNJ 7777120) administered at different disease stages to determine temporal requirements for HRH4 signaling .

For assessing Treg recruitment, perform time-course analyses of tissue infiltration using flow cytometry with comprehensive immune cell panels including CD4+CD25+Foxp3+ Treg markers alongside FITC-conjugated HRH4 antibodies to correlate receptor expression with recruitment patterns . Implement intravital microscopy or adoptive transfer of fluorescently labeled Tregs to directly visualize the migration process in vivo. For mechanistic studies of chemotaxis, perform transwell migration assays comparing wild-type and HRH4-deficient Tregs in response to chemotactic gradients and histamine.

Evaluate Treg function through suppression assays comparing the ability of HRH4-sufficient and HRH4-deficient Tregs to inhibit effector T cell proliferation and cytokine production. Analyze Treg-associated suppressive mechanisms including CTLA-4 expression, IL-10 and TGF-β production, and metabolic competition following HRH4 stimulation or blockade . Determine whether HRH4 signaling influences Treg stability through methylation analysis of the Foxp3 locus and assessment of Foxp3 protein expression stability under inflammatory conditions.

Finally, perform comprehensive immunophenotyping of inflammatory sites, measuring disease-relevant parameters (airway hyperresponsiveness, tissue histology, inflammatory mediators) while simultaneously assessing Treg numbers, HRH4 expression patterns, and correlation with clinical outcomes to establish physiological relevance .

How can researchers address potential cross-reactivity issues when using HRH4 antibodies in tissues expressing multiple histamine receptor subtypes?

Addressing cross-reactivity concerns with HRH4 antibodies requires implementation of rigorous validation procedures, particularly in tissues expressing multiple histamine receptor subtypes (H1R, H2R, H3R, and H4R) that share structural homology . To minimize cross-reactivity issues, start by selecting antibodies raised against unique regions of HRH4 that have minimal sequence homology with other histamine receptors. Antibodies targeting the C-terminal domain (amino acids 204-292) or the third intracellular loop often provide greater specificity than those targeting the transmembrane domains that show higher conservation .

Implement comprehensive negative controls using tissues or cells known to express other histamine receptor subtypes but not HRH4. For example, certain neuronal populations express H3R but not H4R, making them suitable specificity controls. Perform peptide competition assays using peptides corresponding to both the HRH4 immunogen and similar regions from other histamine receptors. Only the HRH4-specific peptide should block antibody binding if the antibody is truly specific .

For critical applications, consider using receptor knockout models or specific siRNA knockdown of HRH4 versus other histamine receptors to validate antibody specificity. When analyzing tissues with multiple histamine receptors, implement dual labeling approaches using antibodies against different histamine receptor subtypes with distinct fluorophores to identify potential co-labeling that might indicate cross-reactivity .

Additionally, complement protein detection with transcript analysis using receptor subtype-specific primers in RT-PCR or RNA in situ hybridization to correlate mRNA expression patterns with antibody labeling, providing an orthogonal validation approach . These comprehensive validation steps will help distinguish genuine HRH4 expression from potential cross-reactivity artifacts.

What strategies can overcome technical challenges when detecting low abundance HRH4 expression in tissue samples?

Detecting low abundance HRH4 expression in tissue samples presents technical challenges that require specialized approaches beyond standard immunostaining protocols. Implement signal amplification techniques such as tyramide signal amplification (TSA), which can enhance FITC signal intensity 10-100 fold without increasing background. This enzymatic amplification method uses HRP-conjugated secondary antibodies to catalyze the deposition of fluorophore-labeled tyramide molecules, significantly boosting detection sensitivity .

Optimize tissue fixation and antigen retrieval methods specifically for HRH4, as overfixation can mask epitopes while insufficient fixation may compromise tissue morphology. Compare multiple antigen retrieval methods (heat-induced epitope retrieval using citrate buffer pH 6.0 versus Tris-EDTA pH 9.0) to determine optimal conditions for HRH4 epitope exposure . Extend primary antibody incubation times to 24-48 hours at 4°C with gentle agitation to enhance antibody penetration and binding, particularly in thicker tissue sections.

Consider alternative detection methods with higher sensitivity than direct FITC conjugates. Implement multi-step detection using unconjugated primary anti-HRH4 antibodies followed by highly-sensitive detection systems such as biotinylated secondary antibodies with streptavidin-conjugated fluorophores or quantum dots that provide superior brightness and photostability compared to conventional fluorophores .

To distinguish specific low-level signal from background, use spectral imaging and linear unmixing techniques that can separate fluorescent signals with overlapping emission spectra from tissue autofluorescence. Additionally, implement tissue clearing techniques such as CLARITY or iDISCO that improve antibody penetration and reduce light scattering in thick tissue sections, enhancing detection of sparse HRH4-expressing cells .

For extremely low abundance detection, consider implementing RNAscope in situ hybridization for HRH4 mRNA as a complementary approach, as this method can reliably detect single mRNA molecules and provides excellent signal-to-noise ratios even for low-copy transcripts .

How should researchers interpret conflicting results between protein-level and mRNA-level HRH4 detection methods?

Interpreting discrepancies between protein-level (immunohistochemistry/Western blot) and mRNA-level (PCR/in situ hybridization) HRH4 detection requires systematic analysis of multiple factors that can contribute to such discordance . First, acknowledge the biological basis for temporal disconnection between transcription and translation. HRH4 mRNA and protein half-lives may differ substantially, leading to scenarios where mRNA is transiently expressed but protein persists, or where post-transcriptional regulation prevents efficient translation despite abundant mRNA.

Scrutinize antibody specificity when protein is detected without corresponding mRNA. Commercial HRH4 antibodies have documented specificity concerns that may lead to false positive protein detection . Validate antibody specificity using the comprehensive approaches outlined in question 3.3, including knockout controls and peptide competition assays. Conversely, when mRNA is detected without corresponding protein, consider whether post-transcriptional regulation mechanisms (microRNAs, RNA-binding proteins) might inhibit translation, or whether the protein undergoes rapid degradation following synthesis.

Examine methodological sensitivity differences between techniques. qRT-PCR can detect very low copy numbers of transcripts that may not produce sufficient protein for immunodetection. Conversely, some antibody-based methods with signal amplification may detect protein levels below the threshold of corresponding mRNA detection by conventional in situ hybridization .

To resolve such discrepancies, implement integrated approaches that simultaneously assess transcription and translation in the same samples. Combine fluorescent in situ hybridization (FISH) for HRH4 mRNA with immunofluorescence for HRH4 protein in the same tissue section to directly correlate transcript and protein at the single-cell level. Additionally, consider using translating ribosome affinity purification (TRAP) to isolate actively translating HRH4 mRNA, bridging the gap between transcriptome and proteome analyses .

Finally, perform careful time-course studies that track both mRNA and protein levels following relevant stimuli (e.g., cytokines, allergens) to establish the temporal relationship between transcription and translation for HRH4 in your specific experimental system.

How might single-cell technologies advance our understanding of HRH4 expression heterogeneity across immune cell populations?

Single-cell technologies offer unprecedented opportunities to unravel the complex heterogeneity of HRH4 expression across diverse immune cell populations, potentially resolving conflicting findings in the literature. Single-cell RNA sequencing (scRNA-seq) can reveal distinct patterns of HRH4 expression across previously unrecognized immune cell subsets, potentially identifying specialized populations with particularly high receptor expression that may respond differentially to histamine signaling . This approach can distinguish HRH4 expression variations between seemingly homogeneous populations such as naïve versus memory T cells, or regulatory T cell subsets with different suppressive mechanisms.

Coupling scRNA-seq with CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) allows simultaneous measurement of HRH4 transcript levels and corresponding surface protein expression using oligonucleotide-labeled antibodies, directly addressing the mRNA-protein correlation at single-cell resolution. This approach can identify post-transcriptional regulation mechanisms specific to certain immune cell subsets .

Single-cell ATAC-seq (Assay for Transposase-Accessible Chromatin) can reveal epigenetic regulation of HRH4 expression by mapping open chromatin regions and transcription factor binding sites controlling receptor expression in different immune cell types. This approach may identify cell type-specific enhancers that drive differential HRH4 expression patterns and potentially explain the elevated expression observed in regulatory T cells compared to conventional T cells .

Mass cytometry (CyTOF) with metal-conjugated anti-HRH4 antibodies enables simultaneous detection of HRH4 alongside dozens of other surface and intracellular markers, providing comprehensive immunophenotyping that can correlate HRH4 expression with functional states across immune cell subsets. Spatial transcriptomics and multiplexed ion beam imaging (MIBI) offer opportunities to map HRH4 expression patterns in complex tissues while preserving spatial context, potentially revealing microanatomical niches where HRH4-expressing cells interact with other immune or structural cells .

These single-cell approaches collectively promise to transform our understanding of HRH4 biology by revealing expression patterns at unprecedented resolution, potentially explaining the seemingly contradictory roles attributed to this receptor in different experimental contexts.

What are the implications of HRH4 polymorphisms and copy number variations for experimental design and clinical translation?

Polymorphisms and copy number variations (CNVs) in the HRH4 gene have significant implications for both experimental design and clinical translation that researchers must carefully consider . Single nucleotide polymorphisms (SNPs) in human HRH4 have been associated with atopic dermatitis and systemic lupus erythematosus, highlighting the receptor's relevance in human inflammatory and autoimmune conditions . When designing experiments using human samples, researchers should implement genotyping strategies to identify common HRH4 polymorphisms, particularly in the coding regions that might affect receptor structure and function or in promoter regions that influence expression levels.

Case-control studies investigating HRH4-targeted therapeutics should stratify participants based on receptor genotype, as functional polymorphisms may predict treatment responsiveness. For in vitro studies using primary human cells, donor genotyping for HRH4 variants will help explain inter-individual variability in receptor expression and function that might otherwise be attributed to experimental factors . When using recombinant expression systems, researchers should ensure they are working with the most common HRH4 allele or alternatively compare multiple variants to assess functional differences.

Copy number variations affecting HRH4 may lead to dosage effects that influence receptor expression levels and downstream signaling intensity. Researchers should implement quantitative PCR methods to assess HRH4 copy number in study participants or experimental cell lines, particularly when working with conditions like systemic lupus erythematosus where CNVs have been reported . For animal studies, recognize that species differences in HRH4 sequence homology and expression patterns may limit direct translation between model organisms and humans. Humanized mouse models expressing human HRH4 variants could provide more translatable insights for therapeutic development.

When developing HRH4-targeted therapeutics, implement pharmacogenomic approaches to identify polymorphisms that affect drug binding or signaling outcomes. This personalized medicine approach may explain variable clinical responses to histamine-modulating therapies and help identify patient subgroups most likely to benefit from HRH4-targeted interventions .

How can computational modeling integrate experimental data to predict HRH4 structure-function relationships and guide targeted therapeutics development?

Computational modeling offers powerful approaches to integrate diverse experimental datasets and predict HRH4 structure-function relationships, ultimately guiding therapeutic development. Modern homology modeling techniques can generate detailed structural models of human HRH4 using crystallographic data from related GPCRs as templates. These models can be refined using molecular dynamics simulations incorporating experimental data on ligand binding, receptor activation, and mutation effects to predict conformational changes associated with different functional states .

Researchers should implement ligand-receptor docking studies to identify crucial binding interactions between HRH4 and both agonists and antagonists. These in silico predictions can guide site-directed mutagenesis experiments to validate key residues involved in ligand recognition and selectivity over other histamine receptor subtypes. For systems-level understanding, agent-based modeling and ordinary differential equation-based approaches can integrate experimental data on HRH4 signaling kinetics to simulate receptor behaviors in complex cellular environments, predicting how changes in receptor density or ligand concentration affect downstream signaling cascades .

Network pharmacology approaches can identify potential off-target effects of existing HRH4-targeted compounds and predict novel applications through drug repurposing. This is particularly valuable given the conflicting pro- and anti-inflammatory effects attributed to HRH4 in different experimental contexts . Machine learning algorithms trained on experimental binding and functional data can predict activity profiles of virtual compound libraries, prioritizing candidates for experimental testing and accelerating the drug discovery process.

To maximize clinical translation potential, physiologically-based pharmacokinetic (PBPK) modeling should integrate tissue distribution data of HRH4 expression with compound pharmacokinetics to predict drug exposure at target sites and optimize dosing regimens. Population pharmacokinetic/pharmacodynamic (PK/PD) models can incorporate data on HRH4 polymorphisms to predict how genetic variations might influence therapeutic responses .

For experimental validation of computational predictions, researchers should implement iterative cycles of in silico modeling followed by targeted experimental testing, with each cycle refining models based on new experimental data. This integrative approach connects computational prediction with experimental validation to systematically advance understanding of HRH4 biology and accelerate the development of precisely targeted therapeutics with optimized efficacy and selectivity profiles .