DCHS1 Antibody, FITC conjugated

What is the optimal application range for DCHS1 Antibody, FITC conjugated?

DCHS1 Antibody, FITC conjugated is primarily optimized for immunofluorescence (IF), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA) applications . The antibody demonstrates strong fluorescence signal when targeting the human DCHS1 protein, particularly in fibroblasts where DCHS1 is predominantly expressed . The conjugation with FITC provides excitation/emission values of 499/515 nm and is compatible with the 488 laser line commonly available in confocal microscopy setups . For optimal results in immunofluorescence applications, dilutions of 1:50-1:100 are typically recommended, though these should be empirically determined for each experimental system .

How should DCHS1 Antibody, FITC conjugated be stored to maintain optimal activity?

DCHS1 Antibody, FITC conjugated should be stored at -20°C for long-term preservation of activity . For short-term usage (within one week), the antibody can be stored at 4°C after thawing . To minimize degradation through freeze-thaw cycles, it is recommended to aliquot the antibody before freezing . Most commercial preparations contain buffer components such as glycerol (40-50%), BSA (0.1-0.5%), and sodium azide (0.01-0.05%) to maintain stability . FITC conjugates are particularly sensitive to photobleaching, so protection from light exposure during storage and handling is essential for maintaining fluorescence intensity .

What controls should be included when using DCHS1 Antibody, FITC conjugated in immunofluorescence studies?

When designing immunofluorescence experiments with DCHS1 Antibody, FITC conjugated, multiple controls should be incorporated:

• Positive tissue control: Human fibroblast cultures or tissue samples known to express DCHS1 should be included .

• Negative tissue control: Melanocytes or keratinocytes, which do not express DCHS1, can serve as biological negative controls .

• Isotype control: A FITC-conjugated rabbit IgG isotype antibody should be used at the same concentration to assess non-specific binding .

• Blocking peptide control: Pre-incubation of the antibody with the immunizing peptide (amino acids 2964-2981 of human DCHS1) should abolish specific staining .

• Autofluorescence control: Unstained samples should be examined to establish baseline cellular autofluorescence in the FITC channel.

These controls help distinguish specific DCHS1 staining from technical artifacts and are essential for publication-quality data .

How can DCHS1 Antibody, FITC conjugated be used to investigate the DCHS1-LIX1L-SEPT9 (DLS) protein complex in cellular systems?

Investigation of the DCHS1-LIX1L-SEPT9 (DLS) protein complex requires sophisticated co-localization approaches. The FITC-conjugated DCHS1 antibody can be effectively combined with differentially labeled antibodies against LIX1L and SEPT9 in multi-color immunofluorescence experiments . Research findings demonstrate that DCHS1 interacts with LIX1L through its cytoplasmic tail (amino acids 2962-3191), while LIX1L simultaneously binds to SEPT9, forming a tripartite complex crucial for DCHS1 stabilization .

For optimal visualization of this complex:

• Perform co-transfection experiments with DCHS1-V5, LIX1L-FLAG, and SEPT9-HA in appropriate cell lines (e.g., HEK293T cells) .

• Use the FITC-conjugated DCHS1 antibody (green channel) alongside spectrally distinct secondary antibodies for detecting the epitope tags of LIX1L and SEPT9.

• Employ confocal microscopy with appropriate channel separation to minimize bleed-through.

• Quantify co-localization using established coefficients (Pearson's, Mander's, etc.).

Notably, biochemical validation through co-immunoprecipitation has confirmed that DCHS1 protein stability is enhanced only in the presence of LIX1L, suggesting that the complete complex formation is necessary for proper DCHS1 function .

What methodological approaches should be used when investigating DCHS1 mutations related to mitral valve prolapse using FITC-conjugated antibodies?

DCHS1 mutations have been identified as causative factors in mitral valve prolapse (MVP) . When investigating these mutations using FITC-conjugated DCHS1 antibodies, researchers should implement the following methodological approach:

• Patient-derived samples: Obtain valve tissue or patient-derived iPSCs from individuals with confirmed DCHS1 mutations.

• 3D culture systems: Establish valve interstitial cell (VIC) cultures or organoids that recapitulate valve development.

• Comparative immunofluorescence: Use DCHS1 Antibody, FITC conjugated at 1:100 dilution to compare subcellular localization between wild-type and mutant DCHS1 .

• Co-staining protocols: Implement dual-labeling with markers of cell migration (e.g., focal adhesion proteins) and extracellular matrix components (e.g., collagens, elastin).

• Live-cell imaging: For dynamic studies, consider using cells transfected with DCHS1-GFP constructs alongside fixed-cell analysis with the FITC-conjugated antibody.

Research has demonstrated that DCHS1 mutations affect cell migration patterns involved in valve formation, leading to mitral valve deformities . The FITC-conjugated antibody allows visualization of altered DCHS1 localization in mutant cells, particularly at cell-cell junctions where DCHS1 normally functions as an adhesion molecule .

How can DCHS1 Antibody, FITC conjugated be utilized in studies of FAT4-DCHS1 signaling in kidney development?

FAT4 and DCHS1 form a ligand-receptor pair with critical functions in kidney development . To investigate this signaling pathway using FITC-conjugated DCHS1 antibodies, researchers should consider:

• Embryonic kidney explant cultures: Utilize ex vivo culture systems of developing kidneys from wild-type, Fat4-/-, or Dchs1-/- mouse models.

• Spatial distribution analysis: Apply DCHS1 Antibody, FITC conjugated (1:100) alongside FAT4 staining to map their expression domains in the developing kidney .

• Quantitative co-localization: Implement high-resolution confocal microscopy with quantitative analysis of protein distribution at tissue boundaries.

• RET signaling assessment: Include phospho-ERK (pERK) staining as a readout of RET signaling activity, which is modulated by FAT4-DCHS1 interaction .

Recent research has revealed that FAT4 and DCHS1 interact to suppress the formation of duplex kidneys by regulating RET signaling . In Fat4-/- mutants, increased levels of pERK indicate enhanced RET signaling, with genetic background influencing the phenotypic outcome . The FITC-conjugated DCHS1 antibody enables precise localization of DCHS1 protein in relation to these signaling events, particularly at the ureteric bud where RET signaling occurs.

What are the most common causes of high background when using DCHS1 Antibody, FITC conjugated, and how can they be addressed?

High background is a frequent challenge when using FITC-conjugated antibodies. For DCHS1 Antibody, FITC conjugated, common causes and solutions include:

For particularly challenging samples, a sequential double blocking strategy may be employed: first with 2% BSA in PBS for 1 hour, followed by 10% normal rabbit serum for 30 minutes before antibody application .

How can researchers optimize protocols when transitioning from unconjugated DCHS1 antibodies to FITC-conjugated versions for multi-color immunofluorescence?

When transitioning from two-step (primary + secondary) to direct detection with FITC-conjugated DCHS1 antibody, several protocol adjustments are necessary:

• Concentration adjustments: FITC-conjugated primary antibodies typically require 2-3 fold higher concentrations than unconjugated versions; start with 1:50 dilution and optimize empirically .

• Incubation modifications:

  • Extend primary incubation time to overnight at 4°C to compensate for potentially reduced avidity after conjugation

  • Include 0.1% Triton X-100 in the antibody dilution buffer to enhance membrane penetration

• Signal amplification options:

  • If signal intensity is insufficient, consider tyramide signal amplification (TSA)

  • Alternatively, anti-FITC antibodies conjugated to brighter fluorophores can be used as a secondary enhancement step

• Photobleaching mitigation:

  • Add anti-fade reagents containing p-phenylenediamine or n-propyl gallate to mounting media

  • Minimize exposure to excitation light during imaging

  • Acquire FITC channel images first in multi-color experiments

• Multiplexing considerations:

  • When combining with red fluorophores, ensure complete spectral separation

  • If using multiple rabbit-derived antibodies, sequential immunostaining with careful blocking between steps is required

What are effective strategies for quantifying DCHS1 expression levels using FITC-conjugated antibodies in heterogeneous tissue samples?

Quantification of DCHS1 expression using FITC-conjugated antibodies requires standardized approaches to account for tissue heterogeneity:

• Standardized image acquisition:

  • Use identical exposure settings across all samples

  • Include calibration beads with known fluorescence intensities

  • Perform sequential imaging of experimental and control samples to minimize session-to-session variability

• Cell-type specific analysis:

  • Implement multi-color immunofluorescence with lineage-specific markers

  • For fibroblast-specific DCHS1 quantification, co-stain with fibroblast markers (vimentin, FSP1)

• Subcellular quantification approaches:

  • For membrane localization: Implement line-scan analysis across cell boundaries

  • For protein complex formation: Perform proximity ligation assay with DCHS1 and interaction partners (LIX1L, SEPT9)

• Normalization strategies:

  • Normalize FITC signal to cell number using nuclear counterstains

  • For tissue sections, calculate DCHS1-positive area relative to total tissue area

  • In cellular contexts, normalize to housekeeping proteins via parallel immunoblotting

• Advanced computational analysis:

  • Implement machine learning-based segmentation to identify cell boundaries

  • Use automated intensity measurement within defined regions of interest

  • Apply batch processing for high-throughput analysis with consistent parameters

How does the performance of DCHS1 Antibody, FITC conjugated compare to other conjugates (HRP, biotin) for different experimental applications?

Different conjugates of DCHS1 antibodies exhibit distinct performance characteristics across applications:

Research has demonstrated that for examining DCHS1's role in mitral valve development, FITC conjugates allow simultaneous visualization of DCHS1 with extracellular matrix components, providing critical spatial information that cannot be achieved with single-color chromogenic methods .

What experimental design considerations should researchers address when investigating DCHS1-FAT4 interactions in tissue development using FITC-conjugated antibodies?

Investigating DCHS1-FAT4 interactions presents unique challenges requiring careful experimental design:

• Trans-interaction considerations:

  • DCHS1 and FAT4 interact in trans across cell membranes of adjacent cells

  • Design co-culture experiments with differentially labeled cell populations expressing either DCHS1 or FAT4

  • Use FITC-conjugated DCHS1 antibody (1:100) alongside a spectrally distinct FAT4 antibody

• Tissue boundary analysis:

  • Focus imaging on tissue boundaries where DCHS1-FAT4 signaling occurs

  • Implement high-resolution imaging techniques (structured illumination, STED) to resolve intercellular interactions

  • Use tissue-clearing techniques (CLARITY, CUBIC) for 3D visualization of interaction domains

• Functional readouts:

  • Include downstream signaling markers (pERK) to assess pathway activation

  • Monitor cell polarization using cytoskeletal markers alongside DCHS1 detection

  • Quantify tissue-specific phenotypes (e.g., kidney branching patterns)

• Genetic manipulation approaches:

  • Compare wild-type, heterozygous, and homozygous mutant tissues

  • Consider mosaic analyses with clonal deletion/overexpression

  • Implement tissue-specific conditional knockout models

Research has established that DCHS1-FAT4 interactions regulate kidney development by modulating RET signaling, with background-specific modifiers influencing phenotypic outcomes . These interactions are conserved from Drosophila to mammals, involving homologs of Dachsous (DCHS1, DCHS2) and Four-jointed (FJX1) .

How should researchers approach species cross-reactivity validation when using DCHS1 Antibody, FITC conjugated in comparative studies?

Validating species cross-reactivity of DCHS1 Antibody, FITC conjugated requires systematic verification across multiple experimental platforms:

• Sequence homology analysis:

  • Compare the immunogen sequence (human DCHS1 amino acids 2964-2981) across target species

  • For example, DCHS1 shows high conservation between human, mouse, and rat in the C-terminal region

• Graduated validation approach:

  • Begin with Western blot verification using tissue lysates from each species

  • Confirm expected molecular weight (~350 kDa) and band pattern

  • Progress to immunofluorescence validation on fixed cells/tissues

  • Include DCHS1 knockout/knockdown controls when available

• Tissue-specific considerations:

  • Test antibody on tissues with known DCHS1 expression patterns

  • For mouse/rat validation, focus on developing heart valves and kidney

  • For human studies, fibroblast cultures provide reliable positive controls

• Technical optimization by species:

  • Adjust fixation protocols based on species (4% PFA for human, 2% PFA for mouse)

  • Optimize antigen retrieval methods independently for each species

  • Determine species-specific optimal antibody concentration

How might DCHS1 Antibody, FITC conjugated be applied in emerging single-cell analysis techniques for developmental biology?

DCHS1 Antibody, FITC conjugated offers significant potential for integration with emerging single-cell technologies:

• Single-cell phenotyping in tissue context:

  • Implement imaging mass cytometry (IMC) with FITC-conjugated DCHS1 antibody for high-parameter analysis

  • Combine with metal-labeled antibodies against developmental markers

  • Achieve simultaneous detection of 40+ proteins while preserving spatial information

• Flow cytometry applications:

  • Develop DCHS1-based cell sorting strategies for isolating specific progenitor populations

  • Implement index sorting to correlate DCHS1 expression with subsequent single-cell transcriptomics

  • Monitor DCHS1 expression changes during differentiation processes

• Spatial transcriptomics integration:

  • Use FITC-conjugated DCHS1 antibody to define regions of interest for spatial transcriptomics

  • Correlate protein localization with gene expression patterns at tissue boundaries

  • Identify transcriptional signatures associated with DCHS1-expressing cells

• Live-cell applications:

  • Explore cell-permeable nanobody-based approaches for intravital imaging

  • Monitor dynamic changes in DCHS1 localization during morphogenetic processes

  • Track cell migration patterns in DCHS1-expressing populations

This integration would enable unprecedented insights into how DCHS1 contributes to cell fate decisions and morphogenetic processes during heart valve formation, kidney development, and other contexts where DCHS1 plays critical roles .

What methodological considerations should be addressed when designing experiments to investigate post-translational modifications of DCHS1 using fluorescently labeled antibodies?

DCHS1 undergoes several post-translational modifications (PTMs), including glycosylation, that affect its function and localization . Investigating these PTMs requires specialized approaches:

• PTM-specific detection strategies:

  • Develop dual labeling protocols combining FITC-conjugated DCHS1 antibody with glycosylation-specific lectins

  • Implement proximity ligation assays to detect specific modified forms

  • Compare total DCHS1 (detected by the FITC-conjugated antibody) with modified subpopulations

• Sample preparation considerations:

  • Preserve PTMs through optimized fixation (avoid methanol for glycosylation studies)

  • Include phosphatase inhibitors for phosphorylation studies

  • Apply mild detergents to maintain membrane integrity while allowing antibody access

• Enzymatic treatment controls:

  • Include deglycosylation controls (PNGase F, O-glycosidase) to verify glycan-dependent signals

  • Implement phosphatase treatments to confirm phosphorylation-specific detection

  • Use these treatments to establish specificity of observed patterns

• Quantification approaches:

  • Develop ratiometric imaging methods comparing modified vs. total DCHS1

  • Implement intensity correlation analysis between DCHS1 and PTM markers

  • Apply machine learning-based classification of subcellular distribution patterns

Understanding DCHS1 PTMs is particularly relevant for heart valve development research, as glycosylation may influence DCHS1-FAT4 interactions and subsequent signaling events critical for proper valve morphogenesis .