HTR7 Antibody, FITC conjugated

What is the HTR7 receptor and why is it a significant research target?

HTR7 (5-HT7) is a G-protein coupled receptor activated by serotonin that plays crucial roles in multiple physiological processes. The signaling cascades activated by HTR7 receptors are involved in circadian rhythm regulation, learning and memory processes, hippocampal signaling, and smooth muscle relaxation in vasculature . Research interest in HTR7 has increased due to its implication in several disorders including autism, neuropsychiatric disorders, epilepsy, and Alzheimer's disease . Additionally, recent findings have established HTR7 as a key mediator of serotonergic itch, making it a potential therapeutic target for chronic itch conditions .

How do FITC-conjugated HTR7 antibodies differ from unconjugated versions?

FITC-conjugated HTR7 antibodies have fluorescein isothiocyanate directly attached to the antibody molecule, providing direct fluorescent detection capabilities without requiring secondary antibodies. The primary advantages include:

• Direct visualization in flow cytometry, immunofluorescence, and live cell imaging

• Reduced background from elimination of secondary antibody cross-reactivity

• Simplified staining protocols with fewer incubation steps

• Capability for direct cell surface detection in live intact cells

What are the typical applications for FITC-conjugated HTR7 antibodies?

FITC-conjugated HTR7 antibodies are particularly valuable for:

• Flow cytometry for detection of HTR7 receptor expression on cell surfaces

• Direct immunofluorescence microscopy in fixed tissues or cells

• Live cell imaging of HTR7 receptor distribution

• Monitoring receptor internalization or trafficking in real-time

• Detecting HTR7 receptor in primary cultures of neurons or other cell types

As demonstrated in validation studies, FITC-conjugated anti-HTR7 antibodies have been successfully used for cell surface detection by direct flow cytometry in various cell types including mouse J774 macrophages and human MEG-01 megakaryoblastic leukemia cells .

How should I design flow cytometry experiments using FITC-conjugated HTR7 antibodies?

For optimal flow cytometry experiments with FITC-conjugated HTR7 antibodies:

• Sample preparation:

  • For adherent cells: gentle enzymatic detachment (avoid trypsin if possible)

  • Maintain cells in cold buffer with sodium azide to prevent receptor internalization

  • Use 1-5×10⁵ cells per sample in 100μl volume

• Staining protocol:

  • Use 2.5-5μg antibody per sample based on cell type (mouse cells: ~2.5μg; human cells: ~5μg)

  • Include proper controls:

    • Unstained cells for autofluorescence

    • Isotype control (Rabbit IgG-FITC) at matching concentration

    • Blocking peptide control for specificity validation

• Instrument settings:

  • Optimize voltages for FITC detection (typically 488nm excitation, 530/30nm emission)

  • Collect sufficient events (minimum 10,000 cells)

  • Compensate for spectral overlap if using multiple fluorophores

• Data analysis:

  • Gate on viable single cells

  • Compare signal intensity to isotype control

  • Quantify as percent positive and/or mean fluorescence intensity

What are the critical factors for successful immunofluorescence using FITC-conjugated HTR7 antibodies?

For optimal immunofluorescence results:

• Fixation considerations:

  • For membrane proteins like HTR7, mild fixation is preferred (2-4% PFA for 10-20 minutes)

  • Overfixation can mask epitopes, particularly for antibodies targeting extracellular domains

• Permeabilization:

  • For antibodies targeting extracellular epitopes (like ASR-037-F which targets N-terminus residues 73-84), permeabilization is unnecessary for surface staining

  • For intracellular epitope detection, use 0.1-0.3% Triton X-100 or 0.1% saponin

• Blocking and antibody incubation:

  • Block with 5-10% normal serum from a species different from antibody source

  • Use antibody at 1:100-1:500 dilution (typically 2-10μg/ml)

  • Incubate 1-2 hours at room temperature or overnight at 4°C

  • Include DAPI for nuclear counterstaining

• Mounting and imaging:

  • Use anti-fade mounting medium to prevent photobleaching

  • Image promptly as FITC is susceptible to photobleaching

  • Store slides at 4°C in the dark

• Controls:

  • Include peptide-blocked antibody control

  • Use tissues from HTR7 knockout animals when available

How can I validate the specificity of FITC-conjugated HTR7 antibodies?

Comprehensive antibody validation should include:

• Genetic controls:

  • Test in HTR7 knockout tissues/cells

  • Compare with HTR7-overexpressing systems (e.g., transfected HEK293 cells)

• Peptide competition:

  • Pre-incubate antibody with immunizing peptide (e.g., CGEQINYGRVEK for extracellular antibodies like ASR-037-F)

  • This should abolish specific staining

• Correlation with gene expression:

  • Compare antibody signal with HTR7 mRNA expression in tissues

  • Use RT-PCR to confirm receptor expression in positive samples

• Multiple antibody validation:

  • Compare results with antibodies targeting different epitopes

  • Cross-validate with non-FITC conjugated HTR7 antibodies

• Signal specificity tests:

  • Expected molecular weight in Western blots (~75 kDa for full protein)

  • Expected subcellular localization (primarily membrane)

  • Expected tissue distribution (brain, vascular tissues, other HTR7-expressing tissues)

How can FITC-conjugated HTR7 antibodies be used to study receptor internalization and trafficking?

To study HTR7 receptor dynamics:

• Live cell imaging setup:

  • Plate cells on glass-bottom dishes

  • Use phenol red-free media supplemented with 25mM HEPES

  • Maintain at 37°C during imaging

• Baseline surface labeling:

  • Label surface receptors with FITC-conjugated HTR7 antibody (1-5μg/ml) for 30 minutes at 4°C

  • Wash thoroughly to remove unbound antibody

• Stimulation protocol:

  • Add HTR7 agonist (e.g., LP44 at 85.7μM) or serotonin (5-HT)

  • Image at regular intervals (1-5 minutes) for 30-60 minutes

• Quantification methods:

  • Measure changes in membrane vs. intracellular fluorescence intensity

  • Track punctate structures indicating internalized receptors

  • Analyze colocalization with endosomal markers

• Controls:

  • Include antagonist pre-treatment (e.g., SB-269970)

  • Use temperature blocks (4°C) to inhibit internalization

  • Include dynamin inhibitors to block endocytosis

This approach allows visualization of receptor dynamics in response to ligand stimulation and can reveal important aspects of HTR7 regulation and signaling.

How do HTR7 splice variants affect antibody binding and experimental design?

The human HTR7 gene produces three splice variants (5-HT7a, 5-HT7b, and 5-HT7d) with different C-terminal domains . This has significant implications:

• Epitope considerations:

  • Antibodies targeting the C-terminus (like those in search result #2) may have splice variant specificity

  • For example, antibody ABIN570980 recognizes only isoform a (NP_000863.1)

  • Extracellular N-terminal antibodies (like FITC-conjugated ASR-037-F) should detect all variants

• Expression patterns:

  • Splice variants show differential tissue expression

  • When interpreting staining patterns, consider which variants may be present

• Experimental design adjustments:

  • For comprehensive detection, use antibodies targeting conserved regions

  • For splice variant discrimination, use specific C-terminal antibodies

  • Consider using RT-PCR with variant-specific primers alongside antibody detection

• Data interpretation:

  • Negative staining might indicate variant-specific expression rather than absence of HTR7

  • Differential staining intensity could reflect variant expression ratios

What are the best practices for resolving discrepancies in HTR7 detection between techniques?

When facing inconsistent results:

• Technique-specific considerations:

  • Flow cytometry: Cell membrane integrity is crucial; dead cells can give false positives

  • IF/IHC: Fixation conditions dramatically affect epitope availability

  • Western blotting: Denaturation may destroy conformation-dependent epitopes

• Epitope accessibility analysis:

  • For FITC-conjugated antibodies targeting extracellular domains (like ASR-037-F):

    • Effective for flow cytometry and surface IF

    • May not work in WB due to denaturation of conformational epitopes

  • For antibodies targeting C-terminus:

    • May require permeabilization for IF/flow cytometry

    • Often more effective in WB applications

• Systematic troubleshooting approach:

  • Test multiple antibody concentrations (titration series)

  • Compare fixation methods (PFA, methanol, acetone)

  • Evaluate antigen retrieval methods for FFPE tissues

  • Compare results in different positive control tissues/cells

• Literature-based validation:

  • Research by Watts et al. (2023) demonstrated that some HTR7 antibodies work well in certain applications but not others

  • Their research showed that antibodies against the C-terminus performed best in Western blots of brain samples, while some antibodies worked better for IHC than WB

How can I minimize background and optimize signal-to-noise ratio with FITC-conjugated HTR7 antibodies?

Background reduction strategies:

• Sample preparation optimization:

  • Fresh samples yield better results than stored samples

  • For tissues: perfusion fixation preferred over immersion fixation

  • Thorough blocking (5-10% serum plus 1% BSA)

  • Include 0.1-0.3% Triton X-100 only if intracellular staining is needed

• Antibody-specific considerations:

  • Titrate antibody to determine optimal concentration

  • Extend washing steps (3-5x 5-minute washes)

  • For flow cytometry, wash cells in protein-containing buffer

  • Pre-clear lysates for Western blotting

• FITC-specific issues:

  • FITC has higher autofluorescence than other fluorophores

  • Use TBS instead of PBS to reduce background

  • Include quenching step (e.g., 50mM NH₄Cl for 15 minutes after fixation)

  • Consider Sudan Black B (0.1-0.3%) to reduce tissue autofluorescence

• Controls for distinguishing true signal:

  • Include isotype control at same concentration

  • Use peptide competition controls

  • Compare with secondary-only controls when using indirect methods

What are the most common reasons for false positive and false negative results with HTR7 antibodies?

False positives may result from:

• Non-specific binding to other serotonin receptors

• Fc receptor binding in immune cells

• Autofluorescence (particularly in FITC channel)

• Cross-reactivity with similar epitopes in other proteins

• Dead/damaged cells in flow cytometry

• Insufficient blocking of endogenous biotin/avidin interactions

False negatives may result from:

• Epitope masking due to fixation or processing

• Receptor internalization or downregulation

• Splice variant expression not recognized by the antibody

• Receptor degradation during sample preparation

• Insufficient antigen retrieval in FFPE tissues

• Photobleaching of FITC fluorophore

Validation controls for both scenarios:

• Include recombinant HTR7-expressing cells as positive controls

• Use brain tissues (particularly hypothalamus) as anatomical positive controls

• Include multiple techniques for cross-validation

• Test antibody in samples with known HTR7 knockout/overexpression

How can receptor density and conformational states affect HTR7 antibody binding?

HTR7, like other GPCRs, exists in dynamic conformational states that can affect antibody recognition:

• Receptor density impacts:

  • Low expression may require signal amplification beyond direct FITC detection

  • High expression can lead to receptor clustering/aggregation affecting epitope accessibility

  • Antibody concentration should be optimized for different expression levels

• Conformational state considerations:

  • Agonist binding (like LP44 or 5-HT) can induce conformational changes

  • G-protein coupling status affects C-terminal epitope accessibility

  • Receptor activation can trigger phosphorylation and internalization

• Experimental approaches to address these issues:

  • Pre-treatment with agonists/antagonists can stabilize specific conformations

  • Membrane preparation methods can affect receptor integrity (detergent selection is critical)

  • For flow cytometry, sodium azide can prevent internalization

  • Crosslinking prior to lysis can preserve protein complexes

• Advanced analysis:

  • Compare staining patterns in presence/absence of HTR7 agonists

  • Use proximity ligation assays to detect protein-protein interactions

  • Consider detergent selectivity for solubilization (mild non-ionic detergents preserve conformations)

How can FITC-conjugated HTR7 antibodies be integrated into multi-parameter immunophenotyping strategies?

For complex immunophenotyping:

• Panel design considerations:

  • FITC emission spectrum (peak ~520nm) requires compensation from PE, APC channels

  • Recommended pairing fluorophores: PE (574nm), PE-Cy7 (785nm), APC (660nm)

  • Avoid tandem dyes that include fluorescein (e.g., FITC-PE)

• Cell type-specific strategies:

  • For neural tissues: combine with NeuN, GFAP, Iba1 to identify HTR7+ cell types

  • For immune cells: combine with CD45, CD11b, etc. to identify HTR7+ immune populations

  • For skin studies: combine with keratinocyte/fibroblast markers to study itch mechanisms

• Advanced flow cytometry applications:

  • Use HTR7-FITC in mass cytometry (CyTOF) panels with metal-conjugated antibodies

  • Integrate with cell sorting for isolation of specific HTR7+ populations

  • Combine with viability dyes and cell cycle markers for functional studies

• Imaging applications:

  • Use in multiplex immunofluorescence with spectral unmixing

  • Combine with fluorescent reporter systems (e.g., calcium indicators) for functional studies

  • Integrate with super-resolution microscopy techniques

What are the emerging applications of HTR7 antibodies in studying receptor-TRP channel interactions?

Recent research has revealed critical HTR7-TRPA1 interactions in itch signaling :

• Co-detection strategies:

  • Use FITC-conjugated HTR7 antibodies with differently labeled TRPA1 antibodies

  • Look for colocalization at membrane/submembrane regions

  • Quantify Pearson's correlation coefficient in different treatment conditions

• Functional coupling analysis:

  • Pre-treatment with HTR7 agonists (LP44) or 5-HT to activate pathways

  • Monitor calcium influx in HTR7+/TRPA1+ neurons

  • Track changes in receptor distribution after activation

• Proximity studies:

  • Use FITC-HTR7 antibodies in proximity ligation assays (PLA) with TRPA1 antibodies

  • Employ FRET techniques to measure nanoscale interactions

  • Utilize super-resolution microscopy to visualize receptor clustering

• Pathophysiological relevance:

  • Apply in mouse models of atopic dermatitis to assess therapeutic potential

  • Study in chronic itch conditions where HTR7-TRPA1 pathway is implicated

  • Analyze receptor expression in human inflammatory skin conditions

This emerging area follows from the discovery that "activation of HTR7 promoted opening of the ion channel TRPA1, which in turn triggered itch behaviors" , suggesting important therapeutic applications for chronic itch conditions.

How can researchers utilize HTR7 antibodies to investigate receptor roles in neuropsychiatric disorders?

For studying HTR7 in neuropsychiatric conditions:

• Brain region-specific analysis:

  • Use FITC-conjugated HTR7 antibodies for high-resolution mapping in:

    • Hypothalamus (implicated in circadian rhythm)

    • Hippocampus (learning and memory)

    • Prefrontal cortex (depression, anxiety)

  • Compare expression patterns between control and disease models

• Neuron subtype characterization:

  • Combine with markers for:

    • Glutamatergic neurons (VGlut1)

    • GABAergic neurons (GAD67)

    • Dopaminergic neurons (TH)

    • Serotonergic neurons (TPH)

  • Identify specific circuits expressing HTR7

• Disease model applications:

  • Autism spectrum disorders: examine receptor expression in relevant mouse models

  • Depression: analyze HTR7 distribution in stress models and post-mortem tissues

  • Alzheimer's disease: investigate relationship to tau pathology

  • Epilepsy: study HTR7 expression changes in seizure models

• Pharmaceutical compound screening:

  • Use antibodies to track receptor expression changes after drug treatment

  • Monitor internalization/trafficking in response to novel therapeutics

  • Combine with functional readouts to correlate receptor modulation with behavior