ITIH1 Antibody, FITC conjugated

What is ITIH1 and what cellular functions does it regulate?

ITIH1, also known as Serum-derived hyaluronan-associated protein (SHAP), functions primarily as a carrier of hyaluronan in serum and as a binding protein between hyaluronan and other matrix proteins. It plays a critical role in regulating the localization, synthesis, and degradation of hyaluronan, processes essential for cells undergoing various biological activities. Additionally, ITIH1 contains peptide regions capable of stimulating a broad spectrum of phagocytotic cells .

Recent research has revealed that ITIH1 may have tumor-suppressive properties, particularly in renal cell carcinoma (RCC), potentially through modulation of the NF-κB signaling pathway .

How do FITC-conjugated antibodies differ from unconjugated ITIH1 antibodies in application?

FITC-conjugated ITIH1 antibodies provide direct fluorescent detection capabilities without requiring secondary antibody incubation steps. While unconjugated antibodies like those mentioned in the search results require additional detection reagents (such as HRP-conjugated secondary antibodies for Western blotting), FITC-conjugated versions enable:

• Direct visualization in fluorescence microscopy

• Flow cytometric analysis without additional staining steps

• Reduced background in multicolor immunofluorescence experiments

• Simplified workflow in time-sensitive experiments

When selecting between conjugated and unconjugated formats, researchers should consider factors including target abundance, experimental design complexity, and signal amplification requirements.

Which epitopes of ITIH1 are typically targeted by commercial antibodies?

Commercial antibodies against ITIH1 target various epitope regions of the protein. Based on the search results, antibodies are available that recognize:

• Amino acids 507-819 (ABIN7436564)

• Amino acids 413-660

• Amino acids 606-635

• Amino acids 410-592

• Full-length ITIH1 protein (ab70048)

The selection of antibody based on epitope region should align with the experimental goals. For instance, studies focusing on specific functional domains of ITIH1 would benefit from domain-specific antibodies, while detection of total ITIH1 might be better served by antibodies recognizing conserved regions or full-length protein.

What are the optimal applications for FITC-conjugated ITIH1 antibodies versus other detection methods?

FITC-conjugated ITIH1 antibodies are particularly valuable in:

• Immunofluorescence microscopy: For subcellular localization studies, especially when examining ITIH1 translocation during signaling events

• Flow cytometry: For quantifying ITIH1 expression levels across cell populations

• Live cell imaging: For tracking dynamic changes in ITIH1 localization

• Multiplexed immunofluorescence: When combined with other fluorophore-conjugated antibodies targeting related proteins

Compared to enzymatic detection methods like HRP, FITC conjugates provide spatial resolution advantages but may offer less signal amplification. For very low abundance targets, researchers might consider signal amplification techniques or alternative detection strategies.

How should researchers validate ITIH1 antibody specificity in their experimental systems?

Validation of ITIH1 antibody specificity should include:

• Positive and negative control samples: Using cells with known ITIH1 expression levels, such as the RCC cell lines (A498, ACHN) versus HK-2 cells described in the literature

• Knockdown/knockout validation: Comparing staining between ITIH1 siRNA-treated cells and control cells, similar to the approach used in research showing >70% knockdown efficiency with specific siRNAs (e.g., si-#1)

• Overexpression controls: Using ITIH1-overexpressing systems, such as cells transfected with pcDNA-ITIH1 as positive controls

• Blocking peptide competition: Pre-incubating the antibody with the immunizing peptide to confirm signal specificity

• Cross-reactivity assessment: Testing the antibody against related proteins, particularly in species with known cross-reactivity (e.g., pig, as noted for some ITIH1 antibodies)

What fixation and permeabilization protocols are recommended for ITIH1 immunofluorescence studies?

For optimal ITIH1 detection in immunofluorescence studies:

Fixation options:

• 4% paraformaldehyde (10-15 minutes, room temperature) for preserving cellular architecture

• Methanol fixation (-20°C, 10 minutes) for enhanced epitope accessibility, particularly for some cytoskeletal-associated proteins

Permeabilization protocols:

• 0.1-0.2% Triton X-100 (10 minutes) for whole-cell permeabilization

• 0.05% saponin for more gentle permeabilization when studying membrane-associated ITIH1

Since ITIH1 can function as both a secreted protein and may associate with cell surfaces through hyaluronan binding , optimization of fixation conditions may be necessary depending on which pool of ITIH1 is being investigated.

How can researchers quantitatively compare ITIH1 expression levels across different experimental conditions?

For quantitative assessment of ITIH1 expression:

• Flow cytometry measurements: Calculate mean fluorescence intensity (MFI) values and compare fold changes between conditions

• Quantitative immunofluorescence microscopy:

  • Establish consistent exposure settings across all samples

  • Measure integrated pixel intensity within defined cellular regions

  • Apply background subtraction algorithms

  • Normalize to cell number or area

• Western blot comparison: For validation of fluorescence-based measurements

  Technique	Advantages	Limitations	Normalization Strategy
  Flow cytometry	Single-cell resolution, high throughput	Limited spatial information	Isotype controls, MESF beads
  Immunofluorescence	Spatial information preserved	Lower throughput, subjective analysis	DAPI signal, cell area
  Western blot	Molecular weight confirmation	Population average, no spatial info	GAPDH or other housekeeping proteins

When analyzing renal cancer cells, it's important to note that the reported ITIH1 expression patterns show discrepancies between tissue samples and cell lines. In TCGA database analyses, ITIH1 was significantly higher in tumor tissues compared to normal tissues, yet lower in RCC cell lines (ACHN, A498, 786-O) compared to HK-2 cells . This highlights the importance of validating findings across different experimental systems.

What strategies can mitigate photobleaching when using FITC-conjugated ITIH1 antibodies in extended imaging sessions?

To minimize photobleaching during extended fluorescence imaging:

• Anti-fade reagents: Use mounting media containing anti-fade compounds like DABCO or proprietary commercial anti-fade solutions

• Oxygen scavenging systems: Incorporate glucose oxidase/catalase systems in live imaging buffers

• Reduced illumination intensity: Use minimum required excitation power

• Minimized exposure times: Balance signal quality with exposure duration

• Sequential acquisition strategies: Limit FITC channel exposures to only when actively acquiring data

• Alternative fluorophores: Consider more photostable alternatives like Alexa Fluor 488 for particularly sensitive applications

When conducting time-lapse experiments to monitor ITIH1 dynamics, researchers should establish photobleaching curves by imaging unstimulated control samples to mathematically correct for signal decay over time.

How can researchers effectively design co-localization studies examining ITIH1 interaction with NF-κB pathway components?

Based on evidence that ITIH1 may regulate the NF-κB pathway in RCC , co-localization studies should:

• Select appropriate fluorophore pairs: Use spectrally distinct fluorophores (e.g., FITC-conjugated ITIH1 antibody and Cy3/Alexa 555-conjugated NF-κB p65 antibody)

• Control for bleed-through: Include single-labeled controls for spectral unmixing

• Optimize fixation conditions: Different fixatives may better preserve specific protein-protein interactions

• Include physiological stimuli: Examine co-localization before and after treatments that activate NF-κB signaling

• Quantify co-localization: Use established metrics such as Pearson's correlation coefficient, Manders' overlap coefficient, or object-based co-localization analysis

• Include protein proximity controls: Consider proximity ligation assays (PLA) for verification of true molecular proximity (<40 nm)

A methodical experimental approach might examine ITIH1 co-localization with multiple NF-κB pathway components including p-NF-κB, IκB, and IKK, which showed altered expression following ITIH1 knockdown in RCC cells .

How should researchers interpret conflicting ITIH1 expression data between cancer tissue samples and cell lines?

The discrepancy observed between ITIH1 expression in RCC tissue samples (higher in tumor vs. normal tissues according to TCGA data) and cell lines (lower in RCC cells vs. normal HK-2 cells) represents an important biological question . When encountering such contradictions, researchers should:

• Consider microenvironmental factors: The different environment of cell growth in vitro versus in vivo might explain expression differences

• Examine cellular heterogeneity: Bulk tumor samples contain multiple cell types, while cell lines represent homogeneous populations

• Assess epigenetic changes: Cell lines may undergo epigenetic drift during prolonged culture

• Verify with multiple methodologies: Combine RNA-seq, qPCR, Western blot, and immunohistochemistry data

• Correlate with clinical parameters: Evaluate how ITIH1 expression relates to patient survival, as higher ITIH1 expression correlated with decreased 5-year survival in RCC patients

These considerations are particularly important when developing ITIH1 as a potential biomarker for disease states or when investigating its functional significance in cancer progression.

What experimental design would best examine the relationship between ITIH1 and the NF-κB pathway using FITC-conjugated antibodies?

To investigate ITIH1's role in NF-κB signaling using fluorescent techniques:

• Dual immunofluorescence study design:

  • Primary staining: FITC-conjugated ITIH1 antibody

  • Secondary staining: Spectrally distinct fluorophore-conjugated antibodies against p-NF-κB, IκB, and IKK

  • Experimental conditions: Control, ITIH1 knockdown, ITIH1 overexpression, with and without NF-κB pathway inhibitor (JSH-23)

• Live-cell imaging approach:

  • Transfect cells with fluorescent protein-tagged ITIH1 and NF-κB components

  • Monitor translocation dynamics following stimulation

  • Compare dynamics in wild-type versus knockdown backgrounds

• Quantitative analysis workflow:

  • Measure nuclear/cytoplasmic ratios of NF-κB components

  • Correlate with ITIH1 expression levels

  • Calculate kinetic parameters of NF-κB activation

This experimental approach builds on findings that ITIH1 knockdown increased phosphorylation levels of NF-κB and reduced IκB while increasing IKK, Cyclin D1, PCNA, and α-SMA expression in RCC cells .

How can researchers address weak or absent signals when using FITC-conjugated ITIH1 antibodies?

When confronting weak or absent signals:

• Epitope masking assessment: Try multiple antibodies targeting different ITIH1 epitopes, such as those recognizing amino acids 507-819, 413-660, or full-length protein

• Antigen retrieval optimization: For fixed tissues or cells, test different antigen retrieval methods (heat-induced, enzymatic, pH variations)

• Antibody concentration titration: Establish optimal antibody concentrations using a dilution series

• Signal amplification strategies: Consider tyramide signal amplification or other fluorescence enhancement techniques

• Target abundance verification: Confirm ITIH1 expression in your experimental system using PCR or Western blotting prior to immunofluorescence attempts

• Alternative detection systems: If FITC photobleaching is problematic, consider more stable fluorophores

The reported variation in ITIH1 expression between different RCC cell lines suggests researchers should verify baseline expression levels in their specific experimental system .

What controls are essential for validating ITIH1 knockdown or overexpression experiments?

When manipulating ITIH1 expression levels:

For knockdown studies:

• Negative control: Non-targeting siRNA (e.g., sequences like 5'-UUCUCCGUACGUGUCACGUTT-3')

• Multiple siRNA sequences: Test at least two different siRNA constructs, as done with si-#1 and si-#2 in previous research

• Knockdown verification: qPCR (using primers like ITIH1 F: 5'-CTGCAGGGTTTCTACAGCCA-3' and R: 5'-CGCTCTCGGAGCAGTTTCTT-3') and Western blot confirmation

• Concentration optimization: Titrate siRNA concentrations (typically 25-100 pmol)

• Time-course assessment: Monitor knockdown duration and stability

For overexpression studies:

• Empty vector control: Use the backbone vector (e.g., pcDNA3.1) without insert

• Expression verification: Both transcript and protein level confirmation

• Functional validation: Confirm expected phenotypic changes (e.g., reduced proliferation and invasion for ITIH1 overexpression in RCC cells)