KRT5 Antibody

What is KRT5 and why is it an important research target?

KRT5 (Keratin 5) is a 62.4 kilodalton protein in humans also known as CK5, DDD, K5, DDD1, EBS2, and keratin, type II cytoskeletal 5. It serves as a crucial marker in tissue characterization and pathological diagnosis, particularly in distinguishing epithelial cell types and carcinomas. KRT5 is especially important in differentiating between adenocarcinomas and squamous cell carcinomas in lung cancer diagnostics, which has significant implications for treatment selection and patient management . KRT5 expression is also observed in basal-like cells that appear during viral pneumonia, forming distinctive scar-like structures that are relevant to understanding disease pathogenesis and tissue repair mechanisms .

What are the most widely used KRT5 antibody clones and how do they differ in performance?

The most commonly used KRT5 antibody clones include D5/16 B4 and XM26 (mouse monoclonal antibodies) and SP27 and EP1601Y (rabbit monoclonal antibodies). These clones show significant differences in analytical sensitivity and specificity:

Clone SP27 demonstrates significantly higher analytical sensitivity compared to other clones but may show positive reactions in up to 25% of adenocarcinomas, potentially reducing its clinical specificity in lung cancer diagnostics . The choice of antibody clone should therefore be guided by the specific research question and the need for sensitivity versus specificity.

What applications are KRT5 antibodies validated for in scientific research?

KRT5 antibodies have been validated for multiple applications across diverse research contexts:

• Western Blot (WB): For protein quantification and molecular weight verification

• Immunocytochemistry (ICC): For cellular localization studies

• Immunofluorescence (IF): For colocalization with other markers

• Immunohistochemistry (IHC): For tissue expression pattern analysis

• Flow Cytometry (FCM): For cell sorting and quantification

• ELISA: For protein detection in solution

The specific application suitability varies by clone and manufacturer. For instance, antibodies like EP1601Y have been validated for WB, FCM, ICC, IF, and IHC-p applications across human, mouse, and rat samples, with over 176 citations supporting their use in published research .

How should researchers optimize KRT5 antibody protocols for immunohistochemistry?

Optimization of KRT5 antibody protocols for immunohistochemistry requires careful consideration of several factors:

• Antibody selection: Choose the appropriate clone based on your specific research question. For high sensitivity in detecting any KRT5 expression, SP27 may be preferred, while D5/16 B4, XM26, or EP1601Y might be better for distinguishing between adenocarcinomas and squamous cell carcinomas due to their higher specificity .

• Antigen retrieval: Most KRT5 antibody protocols require heat-induced epitope retrieval, typically using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0). The optimal method may vary by clone and tissue fixation.

• Antibody dilution: Titration experiments should be performed to determine the optimal antibody concentration that maximizes specific staining while minimizing background.

• Positive and negative controls: Include appropriate controls in each staining run. Squamous cell carcinomas serve as excellent positive controls, while adenocarcinomas (for most clones except SP27) can serve as negative controls .

• Detection system: Choose a detection system with appropriate sensitivity for your application. Polymer-based detection systems often provide excellent results with KRT5 antibodies.

The staining results should be assessed using a standardized scoring system, such as the H-score (0-300), as utilized in comparative studies of KRT5 antibody clones .

What considerations are important when using KRT5 antibodies for lung cancer differential diagnosis?

When using KRT5 antibodies for lung cancer differential diagnosis, researchers should consider:

• Clone selection impact: Clone SP27 shows higher analytical sensitivity but may produce positive reactions in approximately 25% of adenocarcinomas, potentially leading to misclassification. Clones D5/16 B4, XM26, and EP1601Y demonstrate higher specificity for squamous cell carcinomas .

• Staining pattern interpretation: Consider the pattern and intensity of staining. Strong, diffuse positivity is more indicative of squamous differentiation than focal or weak staining, which may be seen in some adenocarcinomas with SP27 .

• Complementary markers: Use KRT5 in combination with other markers. p40 is highly specific for squamous differentiation and complements KRT5 staining. In a comparative study, p40 showed positivity in 97% of squamous cell carcinomas but only 2% of adenocarcinomas .

• IHC assay validation: Validate your IHC assay using known positive and negative controls. Current lung cancer classification standards are based on older clones like D5/16 B4 and XM26, so using newer, more sensitive clones like SP27 may require adjustment of diagnostic cutoffs .

• Correlation with morphology: Always interpret KRT5 staining in conjunction with morphological features and clinical context for accurate diagnosis.

How can researchers validate KRT5 antibody specificity in their experimental systems?

Validating KRT5 antibody specificity requires a multi-faceted approach:

• Multiple antibody clones: Compare results using different KRT5 antibody clones. Concordant results across multiple clones increase confidence in specificity .

• KRT5 mRNA-ISH correlation: Compare protein expression (by IHC) with mRNA expression (by in situ hybridization). A study found that 71% of adenocarcinoma cores showed weak/scattered KRT5 mRNA expression, while all sufficient squamous cell carcinoma cores showed at least moderate KRT5 mRNA expression .

• Knockout/knockdown validation: Use KRT5 knockout or knockdown models as negative controls when available.

• Positive and negative tissue controls: Include tissues known to express KRT5 (e.g., skin, squamous epithelia) and tissues known not to express KRT5 (e.g., certain adenocarcinomas) as controls.

• Western blot confirmation: Perform Western blotting to confirm the antibody detects a protein of the expected molecular weight (approximately 62.4 kDa for KRT5) .

• Peptide competition: Pre-absorb the antibody with the immunizing peptide to confirm binding specificity.

• Cross-reactivity assessment: Test the antibody on tissues from different species if cross-reactivity is claimed by the manufacturer.

How does KRT5 expression relate to pathological processes in viral pneumonia and lung injury?

KRT5 expression plays a critical role in the pathophysiology of viral pneumonia and lung injury:

• Basal-like cell expansion: Severe viral pneumonia can induce rapid expansion of KRT5+ basal-like cells in small airways and alveoli, forming scar-like structures . This represents a pathological repair mechanism that may contribute to long-term sequelae.

• IFN-γ signaling pathway: Research demonstrates that IFN-γ specifically regulates the formation of dysplastic KRT5+ cells induced by influenza A virus (IAV) infection but not by bleomycin-induced injury. Genetic inactivation of interferon receptor 1 (Ifngr1) in lung epithelial cells results in reduced KRT5+ lung area and decreased Krt5 mRNA expression levels following IAV infection .

• JAK/STAT signaling involvement: IFN-γ promotes the transdifferentiation of intrapulmonary p63+ progenitor cells into KRT5+ cells through JAK/STAT signaling. Treatment with JAK1/JAK2 inhibitors (baricitinib, fedratinib) diminishes this effect both in vitro and in vivo .

• YAP coordination: In viral infection models, inflammatory signals coordinate with YAP to regulate KRT5+ cell formation. The expansion of dysplastic KRT5+ cells involves activation of Src (phosphorylated at Tyr416) when these cells migrate into the alveoli .

• Differential mechanisms: The pathways governing KRT5+ cell formation differ between viral and chemical injury models, highlighting the context-specific nature of epithelial repair responses .

What are the key considerations when analyzing contradictory KRT5 antibody staining results?

When faced with contradictory KRT5 antibody staining results, researchers should systematically investigate:

• Antibody clone differences: Different KRT5 antibody clones show variable sensitivity and specificity. For example, clone SP27 demonstrates significantly higher analytical sensitivity but may produce positive results in some adenocarcinomas that other clones would not detect .

• Technical factors: Variations in tissue processing, fixation time, antigen retrieval methods, detection systems, and antibody concentrations can all contribute to discrepant results.

• Epitope accessibility: The accessible epitopes might differ between samples or experimental conditions, affecting antibody binding.

• Non-specific staining patterns: Some clones, like D5/16 B4, can produce granular staining in adenocarcinomas due to Mouse Ascites Golgi (MAG) reaction, which should not be interpreted as specific KRT5 expression .

• KRT5 mRNA-ISH correlation: When antibody results are contradictory, KRT5 mRNA-ISH can provide valuable validation. Studies have shown that some adenocarcinomas express low levels of KRT5 mRNA despite being negative with most KRT5 antibody clones .

• Biological heterogeneity: True biological variation in KRT5 expression can occur, particularly in mixed tumors or those undergoing transdifferentiation.

• Scoring methodology: Standardize scoring systems (such as H-score) and assess both the percentage of positive cells and staining intensity to reduce subjective interpretation variations .

How can KRT5 antibodies be used in multiplexed immunofluorescence studies?

KRT5 antibodies can be effectively incorporated into multiplexed immunofluorescence studies with these approaches:

• Clone selection for multiplexing: Choose KRT5 antibody clones with minimal cross-reactivity to other targets in your panel. Rabbit monoclonal antibodies like EP1601Y or SP27 may offer advantages in certain multiplex panels due to their high specificity and sensitivity .

• Species compatibility: Select antibodies from different host species to facilitate simultaneous detection with species-specific secondary antibodies. KRT5 antibodies are available as both mouse monoclonal (e.g., D5/16 B4, XM26) and rabbit monoclonal (e.g., EP1601Y, SP27) options .

• Sequential staining protocols: For complex panels, sequential staining with appropriate blocking and stripping steps between antibodies can minimize cross-reactivity.

• Spectral unmixing: Use spectral imaging systems to distinguish between fluorophores with overlapping emission spectra, enabling more markers to be included in a single panel.

• Antibody conjugation: Directly conjugated KRT5 antibodies eliminate the need for species-specific secondary antibodies, reducing background and cross-reactivity in multiplex panels.

• Validation strategies: Validate multiplex panels by comparing staining patterns with single-plex controls to ensure antibody performance is not compromised in the multiplex setting.

• Complementary markers: Combine KRT5 with other epithelial markers (e.g., p63, p40), proliferation markers (e.g., Ki-67), or signaling pathway components (e.g., phospho-Src, YAP) to elucidate complex cellular relationships in tissue contexts .

What are common pitfalls when using KRT5 antibodies and how can they be addressed?

Common pitfalls when using KRT5 antibodies include:

• False positive staining: Clone-specific issues can lead to false positives. For example, SP27 may show positive staining in up to 25% of adenocarcinomas that other clones would classify as negative .

  • Solution: Use multiple KRT5 antibody clones and correlate with morphology and other markers.

• Non-specific Mouse Ascites Golgi (MAG) reaction: Clone D5/16 B4 can produce granular staining in adenocarcinomas due to MAG reaction .

  • Solution: Be familiar with this staining pattern and do not misinterpret it as specific KRT5 expression. Consider using rabbit monoclonal alternatives.

• Variable sensitivity between clones: Significant sensitivity differences exist between clones, with SP27 showing much higher sensitivity than others .

  • Solution: Select the antibody clone based on your specific research question—higher sensitivity for detecting any KRT5 expression versus higher specificity for differentiating carcinoma types.

• Inconsistent staining between batches: Lot-to-lot variation can occur in antibody production.

  • Solution: Include consistent positive and negative controls with each staining run and validate new lots against previous lots.

• Suboptimal antigen retrieval: Inadequate or excessive antigen retrieval can affect staining results.

  • Solution: Optimize antigen retrieval conditions for each tissue type and fixation method.

• Discrepancies between protein and mRNA expression: Some adenocarcinomas show KRT5 mRNA expression without detectable protein by IHC with most clones .

  • Solution: Consider using KRT5 mRNA-ISH as a complementary technique when results are ambiguous.

How should researchers validate batch-to-batch consistency of KRT5 antibodies?

To ensure batch-to-batch consistency of KRT5 antibodies, researchers should:

• Establish reference tissues: Create a panel of reference tissues with known KRT5 expression patterns, including positive controls (squamous cell carcinomas) and negative controls (most adenocarcinomas) .

• Standardize staining assessment: Use quantitative scoring methods such as H-score (0-300) to objectively compare staining results between batches .

• Titration experiments: Perform antibody titration with each new lot to determine the optimal concentration that provides consistent results.

• Western blot validation: Confirm consistent detection of the 62.4 kDa KRT5 protein band across different antibody lots .

• Parallel testing: Run the new antibody lot in parallel with the previous lot on identical tissue sections to directly compare performance.

• Document lot-specific characteristics: Maintain detailed records of each antibody lot's performance characteristics, including optimal dilution, staining intensity, and background levels.

• Multi-clone verification: For critical applications, verify results with alternative KRT5 antibody clones to ensure consistency of biological findings independent of antibody lot variations .

• Manufacturer communication: Report significant lot-to-lot variations to the manufacturer and request technical support or replacement if necessary.

What criteria should be used to evaluate the quality of KRT5 antibody staining in tissue sections?

Evaluation of KRT5 antibody staining quality in tissue sections should be based on these criteria:

• Signal-to-noise ratio: High-quality staining should show clear differentiation between KRT5-positive cells and background. Cytoplasmic staining pattern should be clean and well-defined .

• Staining pattern consistency: KRT5 should show cytoplasmic staining in basal epithelial cells and squamous cell carcinomas. The staining pattern should be consistent across similar cell types within the section .

• Positive control performance: Known KRT5-positive tissues (e.g., squamous epithelia, squamous cell carcinomas) should show the expected staining pattern and intensity .

• Negative control performance: Tissues known not to express KRT5 should remain negative. Most adenocarcinomas should be negative with clones D5/16 B4, XM26, and EP1601Y .

• Absence of artifacts: Quality staining should be free from edge artifacts, precipitation deposits, and non-specific nuclear or stromal staining.

• Counterstain quality: Appropriate counterstaining should provide context without obscuring KRT5-specific staining.

• Reproducibility: Consistent staining should be achieved across technical replicates and between runs.

• Correlation with alternate detection methods: High-quality KRT5 staining should correlate with KRT5 mRNA-ISH results, particularly in tissues with high expression levels .

• Gradation of intensity: The staining should demonstrate appropriate gradations of intensity corresponding to different levels of KRT5 expression, allowing for semi-quantitative assessment .

How might novel KRT5 antibody applications advance our understanding of epithelial pathophysiology?

Novel KRT5 antibody applications hold significant potential for advancing epithelial pathophysiology research:

• Single-cell analysis: Combining KRT5 antibodies with single-cell sequencing technologies could reveal heterogeneity within KRT5+ cell populations and identify novel subpopulations with distinct functions in tissue homeostasis and repair .

• Live-cell imaging: Development of non-toxic fluorescently tagged KRT5 antibody fragments would enable real-time tracking of KRT5+ cell dynamics during development, injury response, and disease progression.

• Lineage tracing studies: Using KRT5 antibodies in conjunction with genetic lineage tracing could further elucidate the fate of KRT5+ progenitor cells in different pathological contexts, particularly in understanding their role in post-infection tissue remodeling .

• Therapeutic targeting: KRT5 antibodies conjugated to therapeutic agents could potentially target squamous cell carcinomas with high specificity, especially using highly specific clones.

• Organoid research: KRT5 antibodies can help identify and isolate cells for organoid culture systems, enabling better in vitro modeling of epithelial tissues and diseases .

• Mechanistic pathway studies: Further investigation of the relationship between inflammatory signals (like IFN-γ) and KRT5+ cell formation could reveal novel therapeutic targets for preventing pathological tissue remodeling after viral infections .

• Cross-tissue comparisons: Systematic analysis of KRT5 expression patterns across different epithelial tissues could reveal tissue-specific roles in homeostasis and disease.

What emerging technologies might enhance KRT5 detection sensitivity and specificity?

Emerging technologies that could enhance KRT5 detection include:

• Proximity ligation assays (PLA): This technique could improve specificity by detecting KRT5 only when in proximity to known binding partners, potentially distinguishing functional from non-functional KRT5 expression.

• CODEX multiplexed imaging: This technology allows for the simultaneous detection of dozens of proteins on a single tissue section, enabling comprehensive characterization of KRT5+ cells in their native microenvironment.

• Mass cytometry (CyTOF): Using metal-tagged antibodies and mass spectrometry, this approach could allow for highly multiplexed analysis of KRT5 expression alongside numerous other markers with minimal spectral overlap issues.

• Super-resolution microscopy: Techniques like STORM or PALM could provide nanoscale resolution of KRT5 filament organization in different cell types and disease states.

• Aptamer-based detection: Development of KRT5-specific aptamers might offer advantages in certain applications, potentially providing higher specificity than traditional antibodies.

• Spatial transcriptomics integration: Combining KRT5 antibody detection with spatial transcriptomics could correlate protein expression with the entire transcriptome at specific tissue locations .

• Automated image analysis algorithms: Advanced AI-based image analysis could improve standardization of KRT5 staining interpretation, reducing subjectivity and enhancing reproducibility.

• CRISPR-based protein tagging: Endogenous tagging of KRT5 using CRISPR technology could enable antibody-independent detection strategies with potentially higher specificity.

How might KRT5 antibodies contribute to personalized medicine approaches in oncology?

KRT5 antibodies could make significant contributions to personalized medicine in oncology through:

• Refined tumor classification: More precise characterization of tumors using optimized KRT5 antibody panels could lead to better stratification of patients for targeted therapies, particularly in lung cancer where treatment approaches differ significantly between adenocarcinomas and squamous cell carcinomas .

• Predictive biomarker development: Investigation of KRT5 expression patterns in relation to treatment response could identify predictive biomarkers for specific therapeutic approaches.

• Minimal residual disease detection: Highly sensitive KRT5 antibodies could potentially detect small populations of residual tumor cells after treatment, guiding decisions about adjuvant therapy.

• Circulating tumor cell identification: KRT5 antibodies could help identify and characterize circulating tumor cells of squamous origin, providing a minimally invasive approach to monitor disease progression.

• Liquid biopsy applications: Development of sensitive assays for KRT5 protein fragments in body fluids could potentially serve as biomarkers for squamous cell carcinomas.

• Therapy response monitoring: Tracking changes in KRT5 expression patterns during treatment could provide early indications of therapeutic efficacy or resistance development.

• Combined diagnostic approaches: Integration of KRT5 antibody results with genomic profiling could provide a more comprehensive tumor characterization, enabling truly personalized treatment strategies that consider both phenotypic and genotypic features.

• Immunotherapy patient selection: Research into the relationship between KRT5 expression patterns and tumor immune microenvironment could potentially identify patients more likely to respond to immunotherapeutic approaches.