KRT76 Antibody, Biotin conjugated

What is KRT76 and where is it expressed in normal tissues?

Keratin 76 (KRT76) is an epithelial differentiation marker expressed in the differentiated epithelial layers of the skin, oral cavity, and squamous stomach. Expression begins during embryonic development around day 17.5 (E17.5) in the tongue, palate, and stomach and continues throughout adulthood. In the tongue, KRT76 expression is predominantly observed on the dorsal surface and lateral border, with fewer cells labeled in the ventral tongue. KRT76 is also strongly expressed in the palate and buccal mucosa but not in the outer lip. In all oral epithelia, KRT76 expression is confined to the suprabasal layers .

KRT76 belongs to the type II (basic) keratin family and plays a critical role in maintaining epithelial integrity. Unlike its role in maintaining epidermal barrier function, KRT76 in oral epithelia appears to be dispensable for epithelial integrity but crucial for immune regulation and disease susceptibility .

What is the specificity of the Anti-KRT76 Biotin conjugated antibody (clone AE-3)?

The Anti-KRT76 Mouse Monoclonal Antibody (Biotin conjugated, clone AE-3) recognizes basic (Type II or HMW) cytokeratins, which include specific keratins of varying molecular weights:

• 67 kDa (CK1)

• 64 kDa (CK3)

• 59 kDa (CK4)

• 58 kDa (CK5)

• 56 kDa (CK6)

• 52 kDa (CK8)

This antibody demonstrates broad cross-species reactivity, recognizing KRT76 in multiple species including chicken, cow, dog, human, monkey, mouse, rabbit, and rat. It is a mouse-derived monoclonal antibody of the IgG1 kappa isotype. The clone AE-3 specifically recognizes the 65-67, 64, 59, 58, 56, and 52 kDa keratins of the basic subfamily .

How does KRT76 antibody contribute to cancer diagnosis and research?

KRT76 antibody is a valuable tool in cancer research and tumor diagnosis, particularly as part of broader keratin detection systems. The AE-3 clone is often used in conjunction with clone AE-1 as a broad spectrum anti-pan-keratin antibody cocktail (AE1/AE3), which effectively differentiates epithelial tumors from non-epithelial tumors. This distinction is critical in diagnosing and classifying various malignancies including:

• Squamous cell carcinoma versus adenocarcinoma of the lung

• Liver carcinoma

• Breast cancer

• Esophageal cancer

Downregulation of KRT76 in human oral squamous cell carcinomas (OSCC) correlates with poor prognosis, making the detection of KRT76 expression levels particularly relevant in oral cancer research. Experimental models have shown that loss of KRT76 accelerates carcinogen-induced tumor progression in tissues where it is normally expressed, suggesting its importance as a biomarker for malignant transformation .

What phenotypic changes occur in KRT76-deficient animal models?

KRT76 knockout mice (Krt76-/-) exhibit multiple phenotypic changes that highlight the functional importance of this protein:

• Neonatal skin flaking and hyperpigmentation

• Inflammation and impaired wound healing

• Death prior to 12 weeks of age

• Enlarged spleen (splenomegaly) and lymph nodes (lymphadenopathy)

• Increased numbers of regulatory T cells (Tregs)

• Elevated levels of pro-inflammatory cytokines (IL-6, IL-10, TNFα)

• Increased susceptibility to carcinogen-induced tumors in the tongue and squamous stomach

• Defective tight junctions characterized by mislocalization of claudin-1 (CLDN1)

These phenotypic manifestations indicate that KRT76 plays crucial roles beyond mere structural support, particularly in epithelial barrier function, immune regulation, and tumor suppression .

How does KRT76 interact with tight junction components?

KRT76 has been identified as an essential protein for tight junction (TJ) function through its interaction with claudin-1 (CLDN1), an integral TJ component. Research demonstrates that:

• KRT76 physically interacts with CLDN1 protein

• This interaction is necessary for correct positioning of CLDN1 in tight junctions

• Loss of KRT76 leads to CLDN1 mislocalization, resulting in functionally defective tight junctions

• The mislocalization of CLDN1 has been associated with various dermopathies, including inflammatory diseases like psoriasis

This discovery establishes a previously unknown connection between the intermediate filament cytoskeleton network and tight junctions. This interaction represents a critical link where intermediate filament dysfunction directly influences tight junction formation and function, disrupting epithelial homeostasis .

What is the relationship between KRT76 expression and immune regulation in carcinogenesis?

KRT76 plays a significant immunomodulatory role in oral and gastric cancer development:

• Loss of KRT76 leads to both local and systemic inflammation characterized by:

  • Increased effector T cells (CD4+CD44highCD62Llow) in spleen and lymph nodes

  • Elevated regulatory T cells (Tregs; TCRβ+CD4+CD3+Foxp3+) in lymph nodes and thymus

  • Upregulation of circulating pro-inflammatory cytokines (IL-6, IL-10, TNFα)

  • Increased immune cell infiltration (CD45+ cells) in tongue and squamous stomach

• KRT76-deficient Tregs exhibit:

  • Increased suppressive ability

  • Higher expression of CD39 and CD73

  • Enhanced accumulation in the tumor microenvironment

• During carcinogenesis, KRT76 downregulation:

  • Is observed early in hyperplastic oral epithelium

  • Accelerates 4NQO-induced (4-nitroquinoline N-oxide) tumor progression

  • Enhances accumulation of Tregs in the tumor microenvironment

These findings highlight the complex relationship between epithelial cells and the immune system, where loss of a structural protein can significantly alter immune regulation and promote tumor development .

What methodological considerations are important when using biotin-conjugated KRT76 antibodies?

When working with biotin-conjugated KRT76 antibodies, researchers should consider the following methodological aspects:

• Detection systems:

  • Biotin conjugation enables flexible detection using streptavidin-coupled fluorophores or enzymes

  • Avidin-biotin complexes (ABC) can be used for signal amplification

  • Consider endogenous biotin blocking steps in tissues with high biotin content

• Multiplexing capabilities:

  • Biotin-conjugated antibodies allow sequential or simultaneous detection with other antibodies

  • Compatible with tyramide signal amplification (TSA) for enhanced sensitivity

  • Consider potential cross-reactivity when designing multiplex panels

• Fixation and antigen retrieval:

  • Optimal fixation methods vary by tissue type

  • Heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) is commonly effective

  • Cross-linking fixatives may mask biotin, requiring optimization

• Validation steps:

  • Confirm specificity using KRT76 knockout tissues as negative controls

  • Validate antibody performance in each species of interest

  • Perform appropriate absorption controls when using in tissues with high endogenous biotin

These methodological considerations help ensure reliable and reproducible results when using biotin-conjugated KRT76 antibodies in research applications .

How can KRT76 expression changes be monitored during wound healing processes?

Monitoring KRT76 expression during wound healing requires:

• Temporal analysis:

  • Sampling at multiple time points (0h, 24h, 48h, 72h, 7d, 14d post-wounding)

  • Tracking expression patterns from wound edge to center

  • Correlating with stages of healing (inflammation, proliferation, remodeling)

• Spatial considerations:

  • Monitoring expression gradient from normal to wounded tissue

  • Assessing expression in different epithelial layers

  • Evaluating co-localization with proliferation markers (Ki67, PCNA)

• Methodological approaches:

  • Immunohistochemistry/immunofluorescence with biotin-conjugated KRT76 antibody

  • RT-qPCR for mRNA expression analysis

  • Western blotting for protein quantification

  • In situ hybridization for spatial mRNA detection

• Relevant controls:

  • Comparison with other keratin expression patterns

  • Correlation with tight junction protein localization (especially CLDN1)

  • Assessment of inflammatory infiltrate (CD45+ cells)

Research shows that KRT76 is upregulated during normal wound healing and required for this process. Loss of KRT76 leads to the acquisition and infection of skin wounds which fail to properly resolve over time, indicating its critical role in wound repair mechanisms .

What is the molecular mechanism behind KRT76's tumor suppressor function?

The tumor suppressor function of KRT76 operates through multiple molecular mechanisms:

• Barrier function maintenance:

  • KRT76 interaction with CLDN1 ensures proper tight junction formation

  • Functional tight junctions prevent paracellular permeability

  • Maintenance of tissue compartmentalization limits exposure to carcinogens

• Immune regulation:

  • KRT76 expression influences local cytokine production

  • Loss of KRT76 increases pro-inflammatory cytokines (TNFα, IL-4, TSLP)

  • KRT76 deficiency alters Treg numbers and function, creating a pro-tumorigenic immune environment

• Epithelial differentiation:

  • KRT76 marks differentiated epithelial layers

  • Loss disrupts normal differentiation patterns

  • Aberrant differentiation can lead to hyperplasia and dysplasia

• Carcinogen sensitivity:

  • KRT76-deficient tissues show increased susceptibility to chemical carcinogenesis

  • Accelerated tumor development observed with 4NQO treatment

  • Early downregulation of KRT76 observed in hyperplastic epithelium before tumor formation

These mechanisms collectively contribute to KRT76's role as a tumor suppressor, where its loss creates both cellular and immune microenvironmental conditions favorable to carcinogenesis .

What are the optimal experimental controls when using KRT76 antibody in tissue analysis?

For robust experimental design with KRT76 antibody, researchers should implement:

• Negative controls:

  • KRT76 knockout tissue (gold standard)

  • Isotype-matched control antibody

  • Primary antibody omission

  • Tissues known to lack KRT76 expression (e.g., liver, heart)

• Positive controls:

  • Tissues with well-documented KRT76 expression (oral epithelium, skin)

  • Validation against other detection methods (RNA in situ hybridization)

  • Sequential serial sections with alternative KRT76 antibody clones

• Technical validation:

  • Absorption controls using recombinant KRT76 protein

  • Testing multiple antibody dilutions

  • Comparison of different antigen retrieval methods

  • Cross-validation with genomic data (RNA-seq)

• Experimental validation:

  • Correlation with functional readouts (barrier integrity assays)

  • Co-localization with known interaction partners (CLDN1)

  • Comparison between normal and disease states

  • Antibody performance assessment in multiple fixation conditions

These control measures ensure specificity, sensitivity, and reliability of KRT76 antibody staining results across experimental conditions .

How can KRT76 antibody be integrated into multiplex immunoassays?

Integration of biotin-conjugated KRT76 antibody into multiplex immunoassays involves:

• Panel design strategies:

  • Combine with markers of differentiation (Loricrin, Filaggrin, Involucrin)

  • Include inflammatory markers (CD45, CD3, Foxp3)

  • Add tight junction components (CLDN1, Occludin, ZO-1)

  • Incorporate proliferation markers (Ki67, PCNA)

• Sequential multiplexing approaches:

  • Tyramide signal amplification (TSA) with biotin-conjugated antibody

  • Sequential stripping and reprobing

  • Spectral unmixing with multiple fluorophores

  • Cyclic immunofluorescence methods

• Detection optimization:

  • Strategic fluorophore or chromogen selection to avoid spectral overlap

  • Signal amplification methods for low-abundance targets

  • Chromogenic multiplexing with distinct substrate colors

  • Digital scanning and computational analysis

• Analysis methods:

  • Colocalization quantification

  • Spatial relationship mapping

  • Cell phenotyping in tissue context

  • Quantitative image analysis of expression patterns

These approaches facilitate comprehensive analysis of KRT76 expression in relation to multiple cellular and tissue parameters simultaneously, providing deeper insights into its biological functions .

What tissue preparation protocols yield optimal KRT76 antibody performance?

To achieve optimal KRT76 antibody performance, tissue preparation should consider:

• Fixation options:

  • 10% neutral buffered formalin (12-24 hours)

  • Paraformaldehyde (4%, 4-8 hours)

  • Zinc-based fixatives for better epitope preservation

  • Fresh-frozen sections for sensitive applications

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER):

    • Citrate buffer (pH 6.0), 95-98°C for 20 minutes

    • EDTA buffer (pH 9.0) for alternative retrieval

  • Enzymatic retrieval:

    • Proteinase K (20 μg/ml, 15 minutes at 37°C)

    • Trypsin (0.05%, 15 minutes at 37°C)

• Blocking considerations:

  • Endogenous biotin blocking (especially important for biotin-conjugated antibodies)

  • Endogenous peroxidase quenching (3% H₂O₂, 10 minutes)

  • Protein blocking (5% normal serum matching secondary host)

  • Fc receptor blocking for tissues with immune infiltrates

• Section thickness and processing:

  • Optimal thickness: 4-6 μm for brightfield, 8-10 μm for fluorescence

  • Paraffin removal with xylene or xylene substitutes

  • Complete dehydration and rehydration series

  • Extended washing steps to reduce background

These protocols ensure optimal antibody-antigen interaction while minimizing background and maximizing specific signal for KRT76 detection .

How should researchers interpret heterogeneous KRT76 expression patterns in tumor samples?

When analyzing heterogeneous KRT76 expression in tumors, consider:

• Pattern recognition:

  • Focal loss: Indicates early stage changes or field cancerization

  • Complete loss: Associated with more advanced disease

  • Gradient loss: May reflect differentiation status

  • Mosaic pattern: Suggests clonal tumor evolution

• Clinicopathological correlation:

  • Correlation with tumor grade and stage

  • Association with patient outcomes

  • Relationship to treatment response

  • Comparison with normal adjacent tissue

• Multi-marker analysis:

  • Co-registration with proliferation markers

  • Correlation with other keratins (compensatory expression)

  • Association with tight junction protein expression

  • Relationship to immune infiltration patterns

• Quantitative approaches:

  • H-score calculation (intensity × percentage)

  • Digital image analysis for objective quantification

  • Statistical analysis of heterogeneity indices

  • Spatial distribution mapping

Research shows that even within the same mouse, some 4NQO-induced lesions lose KRT76 expression while others retain it, highlighting the importance of thoroughly analyzing heterogeneity. This heterogeneous expression pattern may have prognostic significance, as KRT76 downregulation in human OSCCs correlates with poor prognosis .

What is the significance of KRT76 expression changes in different disease contexts?

KRT76 expression changes carry different implications across disease contexts:

• Cancer progression:

  Disease Stage	KRT76 Expression Pattern	Significance
  Normal tissue	Strong suprabasal expression	Marker of normal differentiation
  Hyperplasia	Focal downregulation	Early change, potential field effect
  Dysplasia	Progressive loss	Precancerous change
  Carcinoma	Substantial/complete loss	Correlates with poor prognosis

• Inflammatory conditions:

  • KRT76 loss promotes inflammatory cytokine production

  • Creates permissive environment for immune dysregulation

  • Associated with increased regulatory T cell accumulation

  • Contributes to chronic inflammation perpetuation

• Wound healing:

  • Upregulation during normal healing

  • Critical for wound resolution

  • Loss associated with impaired healing

  • Important for restoring barrier function

• Developmental context:

  • First expressed at embryonic day 17.5

  • Marks maturation of epithelial tissues

  • Essential for postnatal survival

  • Contributes to epithelial differentiation program

Understanding these context-specific changes enables more accurate interpretation of KRT76 expression patterns in research and diagnostic applications .

How do KRT76 expression changes correlate with alterations in tight junction proteins?

The relationship between KRT76 and tight junction proteins shows:

• Direct protein interactions:

  • KRT76 physically interacts with CLDN1

  • This interaction is necessary for correct CLDN1 positioning

  • Loss of KRT76 leads to CLDN1 mislocalization

  • Effect appears to be specific to certain epithelia

• Tissue-specific effects:

  Tissue Type	Effect of KRT76 Loss on TJ Proteins	Functional Outcome
  Epidermis	Reduced Claudin expression	Barrier dysfunction
  Oral epithelium	No reduction in Claudin1/3/7	Maintained barrier
  Stomach epithelium	No reduction in Claudin1/3/7	Maintained barrier

• Functional consequences:

  • Despite maintained Claudin expression, KRT76 loss in oral/stomach epithelium affects tumor susceptibility

  • Epidermis shows both reduced Claudin expression and delayed barrier formation

  • Different mechanisms may operate in different epithelial contexts

  • Suggests complex relationship beyond simple expression levels

• Implications for barrier function:

  • Tight junction functionality may be compromised despite normal protein levels

  • Protein localization appears critical for function

  • KRT76-mediated scaffolding supports proper TJ assembly

  • Intermediate filament-TJ interaction represents a novel regulatory mechanism

These findings establish KRT76 as a critical linker between the cytoskeleton and tight junction complexes, with tissue-specific effects on barrier function and disease susceptibility .

What are common technical challenges when working with KRT76 antibody and how can they be addressed?

Researchers may encounter these challenges when using KRT76 antibody:

• Background staining issues:

  • Problem: High non-specific background

  • Solutions:

    • Increase blocking time/concentration

    • Optimize antibody dilution

    • Use specific biotin blocking systems

    • Consider alternative detection methods

• Cross-reactivity concerns:

  • Problem: Unexpected staining patterns

  • Solutions:

    • Validate with KRT76 knockout controls

    • Perform peptide competition assays

    • Compare multiple anti-KRT76 antibody clones

    • Confirm with alternative methods (ISH, Western blot)

• Epitope masking:

  • Problem: False-negative results

  • Solutions:

    • Test multiple antigen retrieval methods

    • Optimize retrieval time and temperature

    • Consider alternative fixation methods

    • Try different antibody clones targeting different epitopes

• Quantification difficulties:

  • Problem: Heterogeneous expression patterns

  • Solutions:

    • Use digital image analysis

    • Establish clear scoring criteria

    • Implement tissue microarrays for standardization

    • Utilize H-scores or automated quantification

These troubleshooting approaches help ensure reliable and reproducible results when working with KRT76 antibody across various experimental conditions .

How can researchers validate KRT76 antibody specificity across different species?

Cross-species validation of KRT76 antibody requires:

• Sequential validation approach:

  • Begin with species with known reactivity (e.g., human, mouse)

  • Expand to phylogenetically related species

  • Verify with positive and negative tissue controls from each species

  • Compare staining patterns with predicted expression sites

• Molecular validation methods:

  • Sequence homology analysis of target epitopes

  • Western blot confirmation in multiple species

  • Immunoprecipitation followed by mass spectrometry

  • Recombinant protein controls from different species

• Technical adaptations:

  • Species-specific antigen retrieval optimization

  • Titration of antibody concentration for each species

  • Adjustment of incubation conditions

  • Species-appropriate detection systems

• Functional correlation:

  • Verification of expected tissue distribution

  • Comparison with mRNA expression data

  • Correlation with physiological or pathological states

  • Assessment of subcellular localization consistency

What are emerging applications of KRT76 antibody in cancer immunotherapy research?

KRT76 antibody shows promise for cancer immunotherapy research:

• Tumor microenvironment characterization:

  • Monitoring KRT76 expression changes during immunotherapy

  • Correlation with immune infiltrate composition

  • Assessment of epithelial-immune cell interactions

  • Potential biomarker for treatment response

• Therapeutic target identification:

  • KRT76-Treg axis as intervention point

  • Restoration of KRT76 expression as therapeutic strategy

  • Targeting pathways affected by KRT76 loss

  • Combination approaches with immune checkpoint inhibitors

• Predictive biomarker development:

  • KRT76 expression status as treatment selection marker

  • Correlation with immunotherapy response patterns

  • Integration into multiplex biomarker panels

  • Longitudinal monitoring during treatment

• Mechanistic understanding:

  • How KRT76 loss modulates anti-tumor immunity

  • Role in creating immunosuppressive microenvironment

  • Impact on antigen presentation and recognition

  • Influence on immunotherapy resistance mechanisms

Given that KRT76 deficiency increases regulatory T cells with enhanced suppressive ability and influences pro-inflammatory cytokine production, targeting this pathway represents a novel approach to modulating the tumor immune microenvironment .

How might advanced imaging techniques enhance KRT76 expression analysis in complex tissues?

Advanced imaging approaches for KRT76 analysis include:

• Multiplexed imaging platforms:

  • Cyclic immunofluorescence (CyCIF)

  • Mass cytometry imaging (MIBI, IMC)

  • Co-detection by indexing (CODEX)

  • Multiplexed ion beam imaging (MIBI)

• 3D tissue analysis:

  • Tissue clearing techniques (CLARITY, iDISCO)

  • Light sheet microscopy for whole-mount imaging

  • Volumetric tissue imaging and analysis

  • 3D reconstruction of KRT76 distribution patterns

• Spatial transcriptomics integration:

  • Correlation of KRT76 protein with mRNA expression

  • Single-cell spatial mapping in tissue context

  • Geo-seq for regional expression analysis

  • Digital spatial profiling techniques

• Live cell imaging applications:

  • Dynamic analysis of KRT76-CLDN1 interactions

  • Real-time monitoring during wound healing

  • Intravital microscopy in animal models

  • FRAP analysis of protein dynamics

These advanced techniques would provide unprecedented insights into the spatial and temporal dynamics of KRT76 expression and its relationship to tissue architecture, disease progression, and treatment response .