RHBDD3 Antibody, FITC conjugated

What is RHBDD3 and what is its biological significance?

RHBDD3, also known as Rhomboid domain-containing protein 3 or C22orf3, is a protein widely expressed in immune cells that functions as a negative regulator of Toll-like receptor 3 (TLR3)-mediated natural killer (NK) cell activation. Research has demonstrated that RHBDD3 plays a critical role in attenuating TLR3-triggered acute inflammation by controlling NK cell activation and accumulation in the liver and disrupting NK cell–Kupffer cell interaction . As a feedback inhibitor, RHBDD3 is selectively upregulated in NK cells upon TLR3 stimulation and subsequently inhibits TLR3-triggered IFN-γ and granzyme B expression . At the molecular level, RHBDD3 interacts with DNAX activation protein of 12 kDa and promotes its degradation, thereby inhibiting MAPK activation in TLR3-triggered NK cells .

What are the basic characteristics of RHBDD3 Antibody, FITC conjugated products?

RHBDD3 Antibody, FITC conjugated products typically share these characteristics:

This information is consistent across multiple vendors including Qtonics, Cusabio, Biorbyt, and Abbexa .

What applications are FITC-conjugated RHBDD3 antibodies suitable for?

FITC-conjugated RHBDD3 antibodies are primarily designed for applications that utilize fluorescence detection, including:

• Flow Cytometry (FCM): For detecting RHBDD3 in single-cell suspensions

• Immunofluorescence (IF): Both on paraffin-embedded tissues (IHC-P) and frozen sections (IHC-F)

• Immunocytochemistry (ICC): For cellular localization studies

• ELISA: Some products are tested for this application

The FITC conjugation eliminates the need for secondary antibodies in fluorescence-based detection methods, simplifying experimental procedures and reducing background signal.

What are the optimal storage conditions for maintaining antibody activity?

For optimal preservation of FITC-conjugated RHBDD3 antibodies:

• Store at -20°C or -80°C upon receipt

• Divide into small aliquots to avoid repeated freeze-thaw cycles

• Protect from light due to the photosensitivity of the FITC fluorophore

• Store in the supplied buffer containing 50% glycerol, which prevents freeze damage

• Never store diluted antibody solutions for extended periods

The presence of preservatives like 0.03% Proclin 300 in the storage buffer helps prevent microbial contamination, while the glycerol component provides cryoprotection .

How should researchers troubleshoot diminished fluorescence intensity?

If experiencing reduced fluorescence signal:

• Check storage conditions – improper storage or excessive freeze-thaw cycles can reduce fluorescence

• Examine exposure to light – FITC is susceptible to photobleaching

• Verify sample preparation – fixation methods can affect epitope accessibility

• Test antibody titration – optimize concentration for your specific application

• Consider autofluorescence – tissue components may contribute background signals

• Use positive controls – compare with known samples expressing RHBDD3

• Examine pH sensitivity – FITC fluorescence is optimal at alkaline pH (7.5-8.5)

For flow cytometry applications specifically, adding bovine serum albumin (0.5-1%) to your staining buffer can help stabilize the antibody and minimize non-specific binding .

What are the recommended working dilutions for different applications?

Optimal working dilutions should be determined empirically, but typical starting ranges include:

These ranges are based on manufacturer recommendations and should be optimized for specific experimental conditions .

How can researchers validate RHBDD3 antibody specificity?

A comprehensive validation approach should include:

• Positive and negative controls: Use tissues/cells known to express or lack RHBDD3

• Knockout validation: Compare staining in wild-type versus RHBDD3-deficient samples

• Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

• Multiple antibody verification: Compare staining patterns with other RHBDD3 antibodies targeting different epitopes

• Western blot correlation: Confirm that band size matches the predicted molecular weight

• Subcellular localization: Verify that staining pattern matches known cellular distribution

Researchers studying RHBDD3 function have used RHBDD3-deficient (RHBDD3−/−) mice as valuable negative controls for antibody validation .

How can researchers investigate RHBDD3's role in immune regulation?

Based on documented research approaches :

• In vitro immune cell activation models:

  • Isolate splenocytes or NK cells from wild-type and RHBDD3-deficient mice

  • Stimulate with poly(I:C) (TLR3 agonist) alone or in combination with IL-12/15

  • Measure cytokine production (IFN-γ, IL-6) by ELISA

  • Assess intracellular expression of granzyme B, perforin, and IFN-γ by flow cytometry

  • Evaluate NK cell cytotoxicity against target cells (e.g., YAC-1)

• In vivo inflammation models:

  • Challenge wild-type and RHBDD3-deficient mice with poly(I:C) and D-GalN

  • Monitor indicators of liver inflammation (serum ALT, AST, cytokines)

  • Perform histopathological analysis of liver tissues

  • Conduct NK cell depletion or adoptive transfer experiments

• Cell-cell interaction studies:

  • Co-culture purified NK cells with accessory cells (dendritic cells, Kupffer cells)

  • Analyze contact-dependent mechanisms using transwell systems

  • Investigate RHBDD3's impact on NK cell-Kupffer cell crosstalk

These methodological approaches have revealed that RHBDD3 inhibits TLR3-triggered NK cell activation in a cell-cell contact-dependent manner involving accessory cells .

What are the considerations for using RHBDD3 antibodies in multiplex immunofluorescence?

When incorporating FITC-conjugated RHBDD3 antibodies into multiplex immunofluorescence:

• Spectral overlap: FITC emission spectrum (peak ~515 nm) may overlap with other green fluorophores; use appropriate compensation

• Panel design: Consider FITC's brightness (medium) when assigning fluorophores to targets based on expression levels

• Autofluorescence mitigation: Use appropriate blocking and quenching methods, particularly for tissues with high autofluorescence

• Sequential staining: For complex panels, consider sequential rather than simultaneous staining

• Antibody cross-reactivity: Validate that antibodies in your panel don't interfere with each other

• Imaging parameters: Optimize exposure times to prevent photobleaching of FITC while capturing adequate signal

• Fluorophore stability: FITC is more susceptible to photobleaching than some newer fluorophores

For flow cytometry applications using the 488 nm laser line, researchers should optimize voltage settings for the FITC channel (typically 525/50 nm bandpass filter) .

How does RHBDD3 interact with DNAX activation protein of 12 kDa, and what are the methodological approaches to study this interaction?

Research has shown that RHBDD3 interacts with DNAX activation protein of 12 kDa (DAP12) and promotes its degradation, thereby inhibiting MAPK activation in TLR3-triggered NK cells . To investigate this molecular interaction:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate RHBDD3 from cell lysates and probe for DAP12 by Western blot

  • Perform reciprocal Co-IP with DAP12 antibodies

  • Include appropriate controls (IgG, lysates from RHBDD3-deficient cells)

• Proximity ligation assay (PLA):

  • Use primary antibodies against RHBDD3 and DAP12

  • Visualize protein-protein interactions in situ with single-molecule resolution

• Protein degradation assays:

  • Assess DAP12 protein levels in wild-type versus RHBDD3-deficient cells

  • Use proteasome inhibitors to determine degradation mechanism

  • Perform pulse-chase experiments to measure DAP12 protein half-life

• Functional validation:

  • Analyze MAPK phosphorylation in response to TLR3 stimulation

  • Compare signaling outcomes in cells expressing wild-type RHBDD3 versus mutant forms

These approaches can elucidate the molecular mechanisms by which RHBDD3 regulates immune cell signaling through DAP12-dependent pathways.

What phenotypic differences are observed in RHBDD3-deficient models, and how can these be methodologically assessed?

Studies with RHBDD3-deficient (RHBDD3−/−) mice have revealed several phenotypic differences that can be methodologically assessed :

• Immune cell distribution:

  • Flow cytometric analysis shows comparable distributions of B cells, T cells, NK cells, and myeloid cells between RHBDD3+/+ and RHBDD3−/− mice, suggesting RHBDD3 does not affect immune cell development

• Cytokine production:

  • RHBDD3−/− splenocytes produce higher levels of IFN-γ and IL-6 upon poly(I:C) stimulation

  • Methodology: ELISA or multiplex cytokine assays on culture supernatants

• NK cell effector functions:

  • RHBDD3−/− NK cells express more granzyme B, perforin, and IFN-γ after poly(I:C) stimulation

  • RHBDD3−/− NK cells demonstrate stronger cytotoxicity against YAC-1 target cells

  • Methodology: Intracellular cytokine staining, flow cytometry, cytotoxicity assays

• Liver inflammation:

  • RHBDD3−/− mice exhibit increased NK cell accumulation in liver after poly(I:C) challenge

  • RHBDD3−/− mice show exaggerated elevation of serum ALT, AST, IFN-γ, and IL-6 after poly(I:C) injection

  • Hepatic pathology reveals significant increases in inflammatory infiltrates and necrosis

  • Methodology: Flow cytometry of liver-infiltrating cells, serum biochemistry, histopathological analysis

• Survival outcomes:

  • RHBDD3−/− mice demonstrate accelerated death in acute inflammation models

  • Methodology: Kaplan-Meier survival analysis

These phenotypic assessments provide valuable insights into RHBDD3's role in regulating immune responses and inflammation.

How does the FITC conjugation affect antibody functionality and what alternatives exist?

FITC conjugation offers several advantages but also has limitations:

Advantages:

• Direct detection without secondary antibodies

• Readily excited by common 488 nm lasers

• Well-established fluorophore with known characteristics

Limitations:

• Moderate brightness compared to newer fluorophores

• Susceptible to photobleaching

• pH sensitivity (optimal at pH 7.5-8.5)

• Potential quenching when conjugated at high densities

Alternatives to consider:

• Alexa Fluor 488: More photostable, less pH-sensitive

• PE (phycoerythrin): Brighter fluorescence for low-abundance targets

• Other conjugates: Various fluorophores are available for different spectral needs

• Lightning-Link® conjugation kits: Allow researchers to conjugate their own antibodies with various fluorophores

When deciding between FITC and alternative conjugates, researchers should consider their specific application requirements, instrumentation capabilities, and experimental design.

What quality control parameters should researchers verify when selecting a RHBDD3 antibody?

When selecting a RHBDD3 antibody, researchers should verify:

• Immunogen specificity: Confirm the immunizing peptide sequence (304-323AA for many commercial antibodies)

• Validation data: Check for flow cytometry histograms, Western blot images, or immunofluorescence micrographs

• Lot-to-lot consistency: Request information on quality control between manufacturing lots

• Species reactivity: Verify reactivity with your species of interest (human, mouse, rat)

• Application validation: Ensure the antibody is tested for your specific application

• Purity assessment: Confirm purification method (typically antigen affinity purified)

• Concentration: Verify antibody concentration for accurate dilution calculations

• Storage buffer composition: Consider buffer compatibility with your experimental system

Requesting technical data sheets and validation reports from manufacturers can provide this essential information.

How can researchers optimize RHBDD3 detection in challenging tissue samples?

For optimal detection in challenging samples:

• Fixation optimization:

  • Compare different fixatives (PFA, methanol, acetone) for epitope preservation

  • Titrate fixation times to balance structural preservation and epitope accessibility

• Antigen retrieval:

  • Test heat-induced epitope retrieval methods (citrate, EDTA, Tris buffers)

  • Optimize pH of retrieval solutions (typically pH 6.0, 8.0, or 9.0)

  • Adjust retrieval times and temperatures

• Permeabilization:

  • Optimize detergent type and concentration (Triton X-100, saponin, Tween-20)

  • Adjust permeabilization time for balanced access and structural preservation

• Blocking enhancements:

  • Use species-specific serum matching the host of secondary antibodies

  • Add bovine serum albumin to reduce non-specific binding

  • Consider specialized blocking reagents for tissues with high endogenous biotin or Fc receptors

• Signal amplification:

  • Employ tyramide signal amplification for low-abundance targets

  • Consider biotin-streptavidin systems for enhanced sensitivity

• Background reduction:

  • Use tissue-specific autofluorescence quenchers

  • Implement appropriate washing steps with optimized buffers

These methodological optimizations can significantly improve RHBDD3 detection in difficult samples while maintaining specificity.