KRT15 Monoclonal Antibody

What is KRT15 and what are its expression patterns in normal tissues?

KRT15 (Cytokeratin 15) is a type I cytoskeletal protein with a molecular weight of approximately 49kDa that plays important roles in epithelial cell differentiation and stem cell maintenance. It exhibits a distinctive tissue distribution pattern:

• Expressed discontinuously in the basal cell layer of adult skin epidermis

• Continuously expressed in the basal layer of fetal skin epidermis and nail

• Present in the outer root sheath above the hair bulb in hair follicles

• Expressed homogeneously across all cell layers of the esophagus and exocervix

• Detected exclusively in the basal cell layer of oral mucosa and skin

• Found in the basal plus the next two layers of suprabasal epithelium of the palate

This specific expression pattern makes KRT15 particularly valuable as a marker for epithelial stem cells and for studying epithelial differentiation processes.

What applications are KRT15 monoclonal antibodies validated for?

KRT15 monoclonal antibodies have been validated for multiple research applications, as summarized in the following table:

Different antibody clones may demonstrate varying performance across these applications, so selection should be based on the specific requirements of your experimental design .

What are the recommended protocols for using KRT15 antibodies in immunohistochemistry?

For optimal immunohistochemical detection of KRT15 in tissues, the following protocol is recommended based on validated methods:

Sample preparation:

• Fix tissue samples appropriately (typically 10% neutral buffered formalin)

• Process and embed in paraffin

• Section at 4-5μm thickness

Antigen retrieval:

• Perform heat-induced epitope retrieval by boiling in pH 6.0 buffer for 10-20 minutes

• Allow 20 minutes cooling time after heating

Staining procedure:

• Apply KRT15 primary antibody at the recommended concentration:

  • AE00281: 1-2μg/ml for 30 minutes at room temperature

  • Other clones: Follow manufacturer's recommendations

• Use appropriate detection system (e.g., HRP polymer)

• Develop with DAB substrate

• Counterstain as needed

This protocol has been successfully used to detect KRT15 in various tissues, including prostate carcinoma and basal cell carcinoma samples, with high specificity and minimal background .

How can I troubleshoot non-specific binding in KRT15 immunostaining?

Non-specific binding can compromise data quality when working with KRT15 antibodies. Consider these methodological approaches to improve specificity:

Antibody optimization:

• Titrate antibody concentration to determine optimal dilution

• For AE00281, the recommended range is 1-3μg/ml; adjust as needed

• For CAB4854, recommended dilutions are 1:500-1:1000 for WB and 1:50-1:200 for IF/ICC

Blocking strategies:

• Extend blocking time (30-60 minutes)

• Use serum from the same species as the secondary antibody

• Consider specialized blocking solutions for difficult tissues

Antigen retrieval adjustments:

• Test different pH conditions (pH 6.0 citrate buffer is standard for KRT15)

• Modify retrieval duration (typically 10-20 minutes followed by 20 minutes cooling)

Technical considerations:

• Ensure adequate washing between steps

• Include appropriate negative controls (omit primary antibody)

• Consider using monoclonal antibodies with demonstrated mono-specificity, such as AE00281, which has been tested against >19,000 full-length human proteins with no cross-reactivity to related keratin proteins

What considerations are important when selecting a KRT15 monoclonal antibody?

When choosing a KRT15 antibody for your research, consider these key factors:

Species reactivity:

• Ensure compatibility with your experimental model:

  • AE00281: Human

  • MAB10723: Human

  • CAB4854: Human, Rat

  • BF0225: Human, Monkey

Antibody validation:

• Review validation data for your specific application

• Check whether the antibody has been tested in tissues/cells similar to your experimental system

• Evaluate evidence of mono-specificity (e.g., AE00281 has been tested against >19,000 human proteins)

Technical specifications:

• Host species (mouse vs. rabbit) may impact compatibility with other antibodies in multiplexing

• Clone type and isotype (e.g., IgG2b for AE00281, IgG for CAB4854)

• Epitope information (when available)

Application-specific performance:

• For IHC, antibodies like AE00281 and MAB10723 have demonstrated strong performance

• For complex applications like multiplexing, consider antibodies with low background

How can KRT15 expression be used to identify epithelial stem cell populations?

KRT15 has emerged as an important marker for epithelial stem cells, particularly in skin and hair follicles. Methodological approaches for leveraging KRT15 in stem cell research include:

Tissue-specific expression analysis:

• The discontinuous expression pattern of KRT15 in adult epidermis correlates with stem cell niches

• KRT15-positive cells in the hair follicle bulge region represent epithelial stem cells with multipotent capabilities

• The continuous expression in fetal epidermis reflects the higher proportion of stem/progenitor cells during development

Co-expression analysis:

• Combine KRT15 staining with other stem cell markers using multicolor immunofluorescence

• Perform sequential sections to correlate KRT15 expression with markers that might share the same host species

• Analyze co-localization using digital image analysis

Functional validation:

• Use flow cytometry with KRT15 antibodies like BF0225 or MAB10723 to isolate KRT15-positive cells

• Assess stem cell properties through colony-forming assays, differentiation studies, and in vivo transplantation experiments

When using KRT15 as a stem cell marker, it's important to consider tissue context and developmental stage, as its expression and correlation with stemness may vary across different epithelial tissues.

What are the critical considerations for dual/multiple immunolabeling with KRT15 antibodies?

Multiple immunolabeling involving KRT15 requires careful experimental design:

Antibody compatibility:

• Choose antibodies from different host species when possible (e.g., mouse anti-KRT15 and rabbit anti-second target)

• Consider antibody isotypes (AE00281 is IgG2b, kappa ; CAB4854 is IgG )

• Verify that the selected KRT15 antibody clone is compatible with your multiplexing method

Staining optimization:

• Determine optimal conditions for each antibody individually before combining

• Test different antigen retrieval methods compatible with all targets

• Extend washing steps to reduce background

Controls for multiplexing:

• Include single-stained controls for each antibody

• Prepare negative controls (omitting each primary antibody)

• Consider absorption controls with blocking peptides where available

Imaging considerations:

• Capture single-channel images separately before merging

• Apply channel bleed-through correction

• Use appropriate co-localization analysis methods

For example, when combining mouse anti-KRT15 (such as AE00281 or BF0225) with other epithelial markers, careful optimization of each step is required to ensure specific detection while avoiding false co-localization signals .

How do different fixation methods affect KRT15 immunoreactivity?

Fixation significantly impacts KRT15 detection:

Formalin fixation:

• Standard method with good preservation of KRT15 antigenicity

• Requires heat-induced epitope retrieval (HIER) for optimal detection

• AE00281 and MAB10723 are validated for use with formalin-fixed, paraffin-embedded (FFPE) tissues

Heat-induced epitope retrieval:

• For FFPE samples, HIER at pH 6.0 for 10-20 minutes is recommended

• Allow 20 minutes cooling time after HIER for optimal epitope renaturation

Alternative fixation methods:

• Methanol or ethanol fixation can preserve KRT15 without requiring antigen retrieval

• Useful for immunofluorescence in cultured cells

• Acetone fixation works well for frozen sections

Comparative analysis:

• When evaluating fixation methods, process identical tissue regions with standardized antibody concentrations

• Quantify immunoreactivity using digital image analysis

• Assess background, signal-to-noise ratio, and specific staining patterns

Understanding these fixation-dependent effects allows researchers to optimize protocols for specific experimental questions when studying KRT15 expression.

What is the emerging role of KRT15 in cancer research, particularly breast cancer?

Recent studies suggest potentially significant roles for KRT15 in cancer biology:

KRT15 in breast cancer progression:

• KRT15 expression may help discriminate invasive breast cancer (IBC) from ductal carcinoma in situ (DCIS), with an area under the curve (AUC) of 0.895 (95% CI = 0.836–0.954)

• Researchers have proposed that KRT15 might have potential value in the early screening of breast cancer

• There appears to be a correlation between KRT15 expression and HER2 status in breast cancer patients, though more comprehensive data are needed

Methodological approaches:

• Immunohistochemical analysis of KRT15 in tissue microarrays to assess expression across cancer stages

• Correlation of KRT15 expression with established markers (e.g., HER2)

• Development of multi-marker panels incorporating KRT15 for improved diagnostic accuracy

Technical considerations:

• Use of monoclonal antibodies (like AE00281, MAB10723) for consistent results across samples

• Standardized IHC protocols with appropriate controls

• Digital pathology approaches for objective quantification

For researchers investigating KRT15 in breast cancer, current evidence suggests it may have diagnostic potential, but more extensive studies are needed to fully establish its clinical utility .

How can quantitative analysis of KRT15 expression be optimized for research studies?

Quantitative analysis of KRT15 expression requires a systematic approach:

Immunohistochemistry optimization:

• Select a KRT15 antibody with proven specificity (e.g., AE00281, which has been tested against >19,000 human proteins)

• Standardize all steps of the IHC protocol:

  • Consistent section thickness

  • Identical antigen retrieval conditions

  • Uniform antibody concentration and incubation time

Image acquisition standardization:

• Use consistent magnification and exposure settings

• Capture images at sufficient resolution to resolve subcellular localization

• Implement color calibration for consistent values across images

Quantification methods:

• Digital image analysis options:

  • H-score method (intensity × percentage of positive cells)

  • Automated positive pixel counting algorithms

  • Machine learning-based approaches for pattern recognition

• Scoring parameters to consider:

  • Staining intensity

  • Percentage of positive cells

  • Subcellular localization

  • Distribution pattern

Validation:

• Validate digital scores against expert assessment

• Calculate inter-observer and intra-observer variability

• Establish cutoff values based on biological significance

By implementing these methodological considerations, researchers can generate robust quantitative data on KRT15 expression, facilitating reliable biomarker assessment and clinical correlations.