ITGA8 Antibody, HRP conjugated

What is ITGA8 and what biological processes does it regulate?

ITGA8 (Integrin alpha-8) is a transmembrane protein that forms the integrin alpha-8/beta-1 heterodimer. This integrin plays crucial roles in kidney development by regulating the recruitment of mesenchymal cells into epithelial structures . In mature tissues, ITGA8 contributes to the regulation of cell proliferation and apoptosis in renal glomerular cells .

Research has demonstrated that ITGA8 functions as an important attenuator of chronic renal fibrosis. When ITGA8 is deficient, mice exhibit more severe renal fibrosis following unilateral ureteral obstruction (UUO), suggesting its protective role in kidney injury models . The protein's molecular weight is approximately 117.5 kilodaltons, and it undergoes post-translational cleavage into heavy and light chains .

How does ITGA8 antibody contribute to renal fibrosis research?

ITGA8 antibodies are valuable tools for investigating mechanisms of renal fibrosis. Studies have shown that ITGA8 deficiency leads to enhanced TGF-β signaling and increased fibroblast activation in obstructive nephropathy models . When working with these antibodies, researchers can:

• Detect changes in ITGA8 expression during disease progression

• Analyze the spatial distribution of ITGA8 in kidney tissue sections

• Investigate relationships between ITGA8 and fibrotic markers

• Examine differences between wild-type and ITGA8-deficient models

Research has revealed that ITGA8 deficiency correlates with increased phospho-SMAD2/3-positive cells and more α-smooth muscle actin-positive cells in the tubulointerstitium after UUO, indicating enhanced TGF-β signaling and myofibroblast activation .

What applications can ITGA8 antibody, HRP conjugated be used for?

• Western blot analysis (0.01-2μg/mL concentration range)

• Immunohistochemistry (5-20μg/mL concentration range)

• Immunocytochemistry (5-20μg/mL concentration range)

• Immunoprecipitation

Each application requires specific optimization of antibody concentration, incubation conditions, and detection methods to achieve optimal signal-to-noise ratio.

What are optimal storage conditions for maintaining ITGA8 antibody activity?

To maintain the activity of ITGA8 antibody, HRP conjugated, proper storage is essential:

• Upon receipt, store at -20°C or -80°C

• Avoid repeated freeze-thaw cycles that can degrade the antibody and reduce specific binding

• The antibody is provided in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative

For working solutions, aliquoting the antibody into single-use volumes before freezing is recommended to prevent degradation from multiple freeze-thaw cycles.

How should I validate ITGA8 antibody specificity in my experimental system?

Rigorous validation of the ITGA8 antibody is crucial for reliable results:

• Positive controls: Use recombinant human ITGA8 protein or tissues known to express ITGA8 (kidney tissue)

• Negative controls: Include samples from ITGA8-knockout models or tissues that don't express ITGA8

• Peptide competition: Pre-incubate the antibody with immunizing peptide (recombinant human integrin alpha-8 protein, particularly amino acids 114-131) to confirm specific binding

• Multiple detection methods: Compare results across different techniques (e.g., western blot, IHC, ELISA)

• Cross-reactivity assessment: Test antibody reactivity across relevant species when working with animal models

What sample preparation methods yield optimal results with ITGA8 antibody?

Proper sample preparation is critical for detecting ITGA8 accurately:

For protein extraction:

• Use buffers containing protease inhibitors to prevent degradation

• Include phosphatase inhibitors when studying phosphorylation-dependent interactions

• Optimize tissue homogenization methods to maximize protein recovery

For tissue sections:

• Use fresh-frozen or properly fixed tissues (avoid overfixation which may mask epitopes)

• For paraffin-embedded sections, optimize antigen retrieval methods

• Consider thickness of sections (typically 4-6μm) for optimal antibody penetration

How does ITGA8 signaling interact with the TGF-β pathway?

ITGA8 plays a complex role in modulating TGF-β pathway activation during fibrotic responses:

• Research indicates that ITGA8 can bind to latent TGF-β, potentially inhibiting its activation

• In ITGA8-deficient mice, phospho-SMAD2/3 levels (indicators of active TGF-β signaling) are significantly increased following UUO compared to wild-type controls

• ITGA8 deficiency is associated with increased expression of latent TGF-β binding protein 1 (LTBP-1), which potentially supports TGF-β activation

The relationship between ITGA8 and TGF-β pathway components is summarized in the following data table from research on wild-type and ITGA8-deficient mice:

Data presented as fold induction (means±SEM). # p< 0.05 in unilateral ureter obstruction (UUO) versus control tissue (co) .

What methodological approaches can detect changes in ITGA8 expression during kidney injury?

Several methodological approaches can be employed to detect alterations in ITGA8 expression during kidney injury:

• Quantitative PCR: For measuring changes in ITGA8 mRNA expression

  • Include appropriate housekeeping genes for normalization

  • Design primers specific to ITGA8 regions unaffected by splicing variants

• Western blotting: For quantifying protein levels

  • Use the HRP-conjugated antibody for direct detection

  • Typical working dilution range: 0.01-2μg/mL

  • Include loading controls (β-actin, GAPDH)

• Immunohistochemistry: For spatial localization analysis

  • Working dilution range: 5-20μg/mL

  • Quantify positive cells using digital image analysis

  • Correlate with markers of fibrosis or inflammation

• Flow cytometry: For cell-specific expression analysis

  • Combine with cell-type specific markers

  • Use appropriate permeabilization for intracellular epitopes

How can I differentiate between ITGA8 expression in distinct kidney cell populations?

To distinguish ITGA8 expression patterns in different renal cell populations:

• Dual immunofluorescence staining:

  • Combine ITGA8 antibody with markers for:

    • Tubular epithelial cells (e.g., E-cadherin, aquaporins)

    • Fibroblasts (e.g., FSP-1, PDGFRβ)

    • Myofibroblasts (α-SMA)

    • Immune cells (CD45, F4/80)

• Cell isolation approaches:

  • Magnetic or flow cytometry-based cell sorting using cell-specific markers

  • Analysis of ITGA8 expression in isolated cell populations

• Single-cell RNA sequencing:

  • Correlate ITGA8 expression with cell-type specific markers

  • Identify new cell populations expressing ITGA8

How can I minimize background when using HRP-conjugated ITGA8 antibody?

High background is a common challenge with HRP-conjugated antibodies. To minimize non-specific signal:

• Optimal blocking:

  • Use 3-5% BSA or 5-10% normal serum from the same species as the secondary antibody

  • Include 0.1-0.3% Triton X-100 or Tween-20 in blocking solutions

  • Block for at least 1 hour at room temperature

• Antibody dilution optimization:

  • Perform titration experiments to determine optimal concentration

  • Start with manufacturer's recommended range (e.g., 0.01-2μg/mL for western blot)

  • Too high antibody concentration increases background

• Washing optimization:

  • Increase number and duration of washes

  • Use PBS-T (PBS with 0.05-0.1% Tween-20) for effective washing

• Endogenous peroxidase quenching:

  • For tissue sections, pretreat with 0.3-3% hydrogen peroxide solution

  • For cells with high peroxidase activity, use peroxidase blocking reagents

What strategies help overcome inconsistent staining patterns with ITGA8 antibody?

Inconsistent staining can undermine research reliability. Address this issue through:

• Standardized sample processing:

  • Use consistent fixation times and conditions

  • Standardize antigen retrieval methods

  • Process all experimental samples in parallel

• Antibody storage and handling:

  • Avoid repeated freeze-thaw cycles

  • Store at recommended temperatures (-20°C or -80°C)

  • Prepare fresh working dilutions for each experiment

• Controls for each experiment:

  • Include positive and negative controls in every staining batch

  • Use internal controls (structures within the sample known to express or lack ITGA8)

• Detection system optimization:

  • Ensure HRP substrate is fresh and properly prepared

  • Standardize development times

  • Consider signal amplification systems for low-abundance targets

How should I quantify ITGA8-positive cells in tissue sections?

Accurate quantification of ITGA8-positive cells requires systematic approaches:

• Representative sampling:

  • Analyze multiple fields (at least 10) per section

  • Examine multiple sections per sample

  • Use systematic random sampling to avoid bias

• Digital image analysis:

  • Capture images using standardized microscope settings

  • Use software (ImageJ, QuPath, etc.) for automated counting

  • Set consistent thresholds for all samples

• Reporting metrics:

  • Calculate percentage of positive cells (positive cells/total cells)

  • Measure staining intensity using optical density

  • Consider semi-quantitative scoring systems (0-3+ scale)

• Statistical analysis:

  • Use appropriate statistical tests based on data distribution

  • Account for multiple comparisons

  • Consider hierarchical analysis when examining multiple fields and sections

How can I integrate ITGA8 expression data with other markers of fibrosis progression?

To gain comprehensive insights into fibrosis mechanisms:

• Correlation analysis:

  • Compare ITGA8 expression with established fibrosis markers (collagen, α-SMA)

  • Correlate with TGF-β pathway components (pSMAD2/3) and regulators (LTBP-1)

  • Examine relationships with inflammatory markers

• Multivariate analysis:

  • Use principal component analysis or cluster analysis to identify patterns

  • Build regression models to determine predictive value of combined markers

• Temporal analysis:

  • Track changes in ITGA8 and related markers across disease progression

  • Determine whether ITGA8 changes precede or follow other markers

The following data table illustrates correlations between ITGA8 and fibrosis-related markers in experimental models:

Data presented as fold induction (means±SEM). # p< 0.05 in unilateral ureter obstruction (UUO) versus control tissue (co) .