HRH4 Antibody, HRP conjugated

What is HRH4 and why is it significant in cancer research?

HRH4 (Histamine Receptor H4) is a G-protein coupled receptor that mediates histamine signals in peripheral tissues. It is significantly expressed in various tissues including human peripheral leukocytes, bone marrow, colon, liver, lung, small intestine, spleen, testis, thymus, tonsil, and trachea . Recent studies indicate that HRH4 plays a crucial role in cell proliferation in both normal and malignant cells. Notably, HRH4 expression is frequently attenuated in colorectal carcinomas, suggesting its potential role as a tumor suppressor. Research has demonstrated that activation of HRH4 causes growth arrest and influences the expression of cell cycle proteins in colorectal cancer cells through a cAMP-dependent pathway .

What are the common applications for HRH4 antibodies in research?

HRH4 antibodies are widely used in various experimental applications including:

• Western Blotting (WB): For detection of HRH4 protein expression levels in tissue or cell lysates

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of HRH4

• Immunohistochemistry on paraffin-embedded tissues (IHC-P): For localization and visualization of HRH4 in tissue sections

These applications are crucial for investigating HRH4 expression patterns in normal versus pathological conditions, particularly in colorectal cancer research where decreased HRH4 expression has been observed .

What is the significance of HRP conjugation in HRH4 antibodies?

HRP (Horseradish Peroxidase) conjugation to antibodies provides a direct detection system that eliminates the need for separate secondary antibody incubation steps. When using HRP-conjugated antibodies, the enzyme catalyzes a colorimetric, chemiluminescent, or fluorescent reaction when exposed to an appropriate substrate. In standard protocols, primary antibodies (like unconjugated HRH4 antibodies) are typically followed by HRP-conjugated secondary antibodies at dilutions around 1:5000 with incubation for 90 minutes at 37°C . Direct HRP conjugation to the primary HRH4 antibody streamlines this process, potentially improving sensitivity and reducing background interference in detection assays.

How should sample preparation differ for detecting HRH4 in colorectal tissue versus cell lines?

For colorectal tissue samples:

• Tissues should be fixed in 10% neutral-buffered formalin and embedded in paraffin for IHC-P applications

• For protein extraction, fresh tissues should be immediately snap-frozen in liquid nitrogen and then homogenized in appropriate lysis buffer containing protease inhibitors

• When comparing normal adjacent tissues (ANTs) with colorectal carcinoma samples, ensure proper case-matching and tissue preservation techniques to maintain protein integrity

For cell lines:

• Cultured cells (such as Lovo or CACO-2 cell lines commonly used in HRH4 research) should be harvested at 70-80% confluence

• For western blotting, lyse cells directly in a buffer containing detergents like NP-40 or RIPA with protease inhibitors

• For immunofluorescence, cells can be grown on coverslips, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100

The key difference lies in tissue processing techniques, as tissues require more extensive preparation to preserve architecture while ensuring antigen accessibility.

What optimization strategies should be considered when using HRH4 antibodies for Western blotting?

When optimizing Western blot protocols for HRH4 detection:

• Antibody dilution: Start with the recommended range of 1:300-1:5000 and adjust as needed based on signal strength and background

• Protein loading: 20-40μg of total protein is typically sufficient for HRH4 detection

• Blocking solution: 5% non-fat dry milk or BSA in TBS-T (0.1% Tween-20) is recommended for minimal background

• Membrane type: PVDF membranes generally provide better protein retention and lower background than nitrocellulose for HRH4 detection

• Enhanced chemiluminescence (ECL) substrate selection: Use high-sensitivity substrates for low abundance detection, which is especially important since HRH4 expression may be significantly reduced in cancer samples

• Exposure time optimization: Begin with short exposures (30 seconds) and increase as needed to avoid signal saturation

• Stripping and reprobing considerations: Gentle stripping methods are recommended if multiple proteins are to be analyzed from the same membrane

How can researchers address the challenge of decreased HRH4 expression in colorectal cancer samples?

The attenuated expression of HRH4 in colorectal carcinomas presents a challenge for detection and functional studies. Researchers can address this through several approaches:

• Enhanced detection methods:

  • Use signal amplification techniques such as tyramide signal amplification (TSA)

  • Employ more sensitive substrates for HRP detection

  • Consider using antibody cocktails targeting different epitopes of HRH4

• Experimental controls:

  • Include positive controls with known HRH4 expression (e.g., normal colon tissue)

  • Use H4R-transfected cell lines (like H4R-Lovo cells) as standards for comparison

• Functional assessment strategies:

  • Design experiments that compare HRH4-overexpressing cells with control cells

  • Utilize histamine or clozapine (HRH4 agonist) stimulation to evaluate functional responses

  • Employ cAMP assays to measure downstream signaling of HRH4 activation

• RNA analysis in parallel:

  • Complement protein detection with mRNA analysis using RT-PCR or qPCR

  • Compare protein and mRNA levels to identify potential post-transcriptional regulation mechanisms

What controls should be included when validating HRH4 antibody specificity?

Proper validation of HRH4 antibody specificity is crucial for reliable research outcomes. Essential controls include:

• Positive tissue controls:

  • Normal colon tissue sections with known HRH4 expression

  • Peripheral blood mononuclear cells that naturally express HRH4

  • Cells transfected with HRH4 expression vectors (e.g., H4R-Lovo cells)

• Negative controls:

  • Primary antibody omission control

  • Isotype control antibody (rabbit IgG at equivalent concentration)

  • Tissues or cells with confirmed low or absent HRH4 expression

• Blocking peptide validation:

  • Pre-incubation of antibody with immunizing peptide to demonstrate signal specificity

  • Gradient peptide blocking to establish affinity characteristics

• Multiple detection methods comparison:

  • Cross-validate results using different detection techniques (WB, IHC, IF)

  • Compare staining patterns between different HRH4 antibodies targeting distinct epitopes

• Genetic validation:

  • Use siRNA knockdown or CRISPR/Cas9 knockout systems to confirm signal specificity

  • Test antibody on tissues from HRH4 knockout animal models if available

How should researchers interpret discrepancies in HRH4 expression levels between different detection methods?

Discrepancies in HRH4 expression when using different detection methods may arise from several factors:

• Epitope accessibility differences:

  • Protein conformation changes during various preparation methods may affect antibody binding

  • Fixation protocols in IHC-P may mask certain epitopes that are accessible in WB after denaturation

• Detection sensitivity thresholds:

  • Western blotting may detect lower expression levels than IHC in samples with attenuated HRH4 expression

  • mRNA analysis may show transcript presence while protein detection fails due to post-transcriptional regulation

• Resolution of analysis:

  • IHC provides spatial information but may lack quantitative precision

  • WB offers quantitative assessment but lacks spatial resolution

  • Flow cytometry provides single-cell resolution but may require different antibody properties

• Recommended analytical approach:

  • Employ multiple detection methods for comprehensive analysis

  • Use quantitative techniques (densitometry for WB, digital image analysis for IHC)

  • Report discrepancies transparently with potential explanations

  • Consider cell-type specific expression patterns, especially in heterogeneous tissues

What considerations are important when analyzing HRH4-mediated effects on cell cycle regulation?

When investigating HRH4's role in cell cycle regulation, researchers should consider:

• Experimental design parameters:

  • Cell synchronization protocols to ensure uniform cell cycle stage

  • Appropriate timepoints for analysis (24h, 48h, 72h) following HRH4 stimulation

  • Concentration-dependent effects of histamine or synthetic HRH4 agonists like clozapine

• Key cell cycle proteins to monitor:

  • Cyclin D1 and Cdk2 (typically downregulated upon HRH4 activation)

  • p21^Cip1 and p27^Kip1 (typically upregulated following HRH4 stimulation)

  • These markers provide mechanistic insights into G1 arrest following HRH4 activation

• Downstream signaling pathway analysis:

  • cAMP levels (decreased with HRH4 activation)

  • PKA activity assessment

  • Effects of cAMP modulators (forskolin, Rp-8-Br-cAMPS) on reversing HRH4-mediated effects

• Cell-type specific responses:

  • Different colorectal cell lines may show varying magnitude of response

  • Endogenous HRH4 expression levels influence response intensity

How does HRH4 activation influence chemosensitivity in colorectal cancer models?

Recent research indicates that HRH4 activation can enhance the efficacy of chemotherapeutic agents in colorectal cancer models:

• Enhanced apoptotic response:

  • HRH4 activation promotes 5-Fu-induced cell apoptosis in HRH4-positive colorectal cells

  • This suggests potential combination therapy opportunities

• Mechanism considerations:

  • HRH4 may counteract anti-apoptotic signaling pathways like NF-kappaB

  • cAMP-PKA pathway involvement has been observed in HRH4 activation-mediated cell death in other cell types

• Experimental approaches to study chemosensitization:

  • Combination treatment studies with HRH4 agonists and standard chemotherapeutics

  • Analysis of apoptotic markers (cleaved caspases, PARP cleavage) following combination treatment

  • Dose-response studies to identify optimal synergistic concentrations

• Future research opportunities:

  • Development of colorectal cancer-specific HRH4 targeting strategies

  • In vivo studies using HRH4 knockout mice to validate chemosensitization effects

  • Investigation of potential resistance mechanisms that might develop

What methodological approaches can resolve contradictions in the literature regarding HRH4 expression in colorectal tissues?

The literature contains some contradictions regarding HRH4 expression in colorectal tissues. To address these discrepancies, researchers should consider:

• Standardized tissue collection and processing:

  • Use consistent fixation protocols across studies

  • Implement standardized antigen retrieval methods

  • Document precise anatomical locations of tissue sampling

• Antibody validation and standardization:

  • Employ multiple antibodies targeting different epitopes

  • Provide complete validation data for antibodies used

  • Standardize dilution and incubation conditions between studies

• Comprehensive expression analysis:

  • Combine protein and mRNA detection methods

  • Implement single-cell analysis techniques to detect cell-type specific expression

  • Use quantitative methods with appropriate statistical analysis

• Clinical sample considerations:

  • Account for patient demographics, tumor stage, and previous treatments

  • Include larger sample sizes with appropriate controls

  • Consider tumor heterogeneity by analyzing multiple regions within tumors

• Functional validation:

  • Complement expression studies with functional assays

  • Use genetic manipulation of HRH4 expression to confirm functional relationships

  • Develop organoid models that better recapitulate in vivo conditions

What is the optimal protocol for immunohistochemical detection of HRH4 in paraffin-embedded tissues?

For optimal immunohistochemical detection of HRH4 in paraffin-embedded tissues:

• Tissue preparation:

  • Fix tissues in 10% neutral buffered formalin for 24 hours

  • Process and embed in paraffin using standard protocols

  • Section tissues at 4-5 μm thickness onto positively charged slides

• Deparaffinization and rehydration:

  • Xylene: 3 changes, 5 minutes each

  • 100% ethanol: 2 changes, 3 minutes each

  • 95%, 80%, 70% ethanol: 3 minutes each

  • Distilled water: 5 minutes

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • Pressure cooker method: 3 minutes at full pressure

  • Allow slides to cool in buffer for 20 minutes

• Blocking and antibody incubation:

  • Block endogenous peroxidase: 3% H₂O₂ for 10 minutes

  • Protein blocking: 5% normal goat serum for 1 hour

  • Primary antibody: HRH4 antibody at 1:200-400 dilution, overnight at 4°C

  • Secondary antibody: HRP-conjugated anti-rabbit IgG for 1 hour at room temperature

• Visualization and counterstaining:

  • DAB substrate: 5-10 minutes (monitor microscopically)

  • Counterstain: Mayer's hematoxylin for 30 seconds

  • Blueing: 0.2% ammonia water, briefly

  • Dehydration and mounting with permanent medium

• Controls to include:

  • Positive control: Normal colonic mucosa

  • Negative control: Primary antibody omission

  • Internal control: Compare epithelial cells with stromal components

How should protocols be modified when using directly HRP-conjugated HRH4 antibodies compared to unconjugated primary antibodies?

When transitioning from a two-step (primary + HRP-conjugated secondary) to a direct HRP-conjugated HRH4 antibody system:

• Dilution optimization:

  • Start with higher concentrations than used for unconjugated antibodies

  • Perform titration experiments (typically 1:50 to 1:500 range)

  • Optimize for signal-to-noise ratio rather than absolute signal intensity

• Incubation modifications:

  • Shorter incubation times are typically sufficient (1-2 hours at room temperature rather than overnight)

  • Temperature optimization may be necessary (4°C, room temperature, or 37°C)

  • Consider adding protein carriers (0.1-0.5% BSA) to preserve enzyme activity

• Blocking adjustments:

  • Use blocking solutions compatible with HRP activity

  • Consider specialized blocking reagents to reduce background

  • Include additional blocking steps to prevent non-specific binding

• Signal development considerations:

  • Use substrate optimized for directly-conjugated antibodies

  • Shorter substrate incubation times may be needed

  • Monitor signal development closely to prevent overdevelopment

• Troubleshooting potential issues:

  • Higher background: Increase blocking stringency or dilution

  • Weak signal: Consider signal amplification systems

  • Non-specific binding: Add detergents or increase wash stringency

What experimental approaches are most effective for studying the cAMP/PKA pathway in HRH4-mediated cell cycle regulation?

To effectively study cAMP/PKA pathway involvement in HRH4-mediated cell cycle regulation:

• cAMP level measurement:

  • Use competitive ELISA-based cAMP assays following HRH4 activation

  • Implement real-time cAMP monitoring using FRET-based biosensors

  • Compare forskolin-induced cAMP levels with and without HRH4 agonists

• PKA activity assessment:

  • Use phospho-specific antibodies against PKA substrates (CREB phosphorylation)

  • Employ fluorescent PKA activity reporters in live cells

  • Conduct in vitro kinase assays with immunoprecipitated PKA

• Pharmacological modulators:

  • HRH4 agonists: Histamine, clozapine

  • cAMP modulators: Forskolin (adenylyl cyclase activator), Rp-8-Br-cAMPS (cAMP antagonist)

  • PKA inhibitors: H-89, PKI peptide

• Genetic approaches:

  • siRNA knockdown of PKA catalytic or regulatory subunits

  • Expression of dominant-negative PKA constructs

  • CRISPR/Cas9 targeting of pathway components

• Downstream effector analysis:

  • Monitor cell cycle proteins (cyclin D1, Cdk2, p21^Cip1, p27^Kip1)

  • Assess cell cycle distribution using flow cytometry

  • Analyze colony formation capacity for long-term effects

How can researchers distinguish between HRH4-specific and non-specific effects when using histamine as a stimulus?

Histamine can activate multiple histamine receptor subtypes (H1R-H4R), making it challenging to attribute effects specifically to HRH4. To overcome this:

• Pharmacological approach:

  • Use HRH4-specific agonists (e.g., clozapine at appropriate concentrations)

  • Employ selective antagonists to block specific receptor subtypes

  • Create a pharmacological profile using multiple agonists/antagonists with different selectivity profiles

• Genetic strategy:

  • Compare effects in cells with normal vs. overexpressed HRH4 (e.g., Mock-Lovo vs. H4R-Lovo cells)

  • Use siRNA or CRISPR/Cas9 to specifically knockdown HRH4

  • Rescue experiments by reintroducing wild-type or mutant HRH4

• Signaling pathway verification:

  • Monitor HRH4-specific signaling (cAMP suppression) in parallel with cellular effects

  • Use PTX (pertussis toxin) to inhibit Gαi/o proteins coupled to HRH4

  • Compare calcium mobilization (H1R/H2R-mediated) vs. cAMP suppression (HRH4-mediated)

• Control experimental design:

  • Include cells lacking HRH4 expression as negative controls

  • Use concentration-response curves to identify receptor-specific thresholds

  • Perform time-course studies to distinguish immediate vs. delayed effects

How can HRH4 antibodies be utilized to study the relationship between HRH4 expression levels and colorectal cancer progression?

To investigate the relationship between HRH4 expression and colorectal cancer progression:

• Clinical specimen analysis:

  • Perform IHC on tissue microarrays containing samples from different cancer stages

  • Quantify HRH4 expression using digital image analysis

  • Correlate expression with clinicopathological parameters and patient outcomes

• Comparative expression analysis:

  • Compare HRH4 levels in early vs. advanced CRCs using Western blotting and qPCR

  • Analyze matched samples (tumor and adjacent normal tissue) from the same patients

  • Evaluate expression in premalignant lesions to determine when HRH4 downregulation occurs

• Functional studies in cell models:

  • Generate cell lines with varying levels of HRH4 expression

  • Assess proliferation, migration, invasion, and apoptosis characteristics

  • Evaluate response to histamine in the tumor microenvironment

• In vivo models:

  • Develop xenograft models with HRH4-modulated cell lines

  • Use patient-derived xenografts to maintain tumor heterogeneity

  • Consider HRH4 knockout mouse models for spontaneous tumor development studies

What are the recommended approaches for studying HRH4-mediated enhancement of chemotherapeutic efficacy?

To investigate HRH4's role in enhancing chemotherapeutic efficacy:

• In vitro drug sensitivity testing:

  • Combine HRH4 agonists with chemotherapeutic agents (e.g., 5-Fu)

  • Use multiple colorectal cancer cell lines with varying HRH4 expression

  • Perform dose-response studies to identify optimal concentrations and sequence of administration

• Apoptosis and cell death assessment:

  • Analyze apoptotic markers (Annexin V/PI staining, caspase activation)

  • Evaluate cell death mechanisms (apoptosis, necrosis, autophagy)

  • Monitor mitochondrial membrane potential changes and cytochrome c release

• Signaling pathway analysis:

  • Investigate convergence points between HRH4 and chemotherapy-induced pathways

  • Examine NF-κB signaling, which HRH4 may counteract

  • Assess DNA damage response pathways when combining treatments

• Translational research approaches:

  • Test combinations in patient-derived organoids

  • Develop predictive biomarkers for response to combination therapy

  • Design preclinical studies in animal models to validate in vitro findings