KRT76 Antibody

What is KRT76 and what is its significance in epithelial tissues?

KRT76 (Keratin 76) is a type II intermediate filament protein primarily expressed in the differentiated, non-proliferative layers of specific stratified epithelia in humans and mice. It plays a crucial role in maintaining epithelial integrity in the skin, oral cavity, and squamous stomach . Unlike other keratins that primarily serve as cytoskeletal scaffolds, KRT76 also functions as an important regulator of epithelial immunity and inflammatory responses. The protein's expression is highly tissue-specific, making it an important marker for differentiated epithelial cells in these tissues .

Methodologically, when studying KRT76 expression patterns, researchers should employ immunohistochemistry with validated antibodies on properly fixed tissue samples, complemented by RT-qPCR to quantify expression levels. For co-expression studies, dual immunofluorescence staining with differentiation markers such as Loricrin can provide valuable insights into the relationship between KRT76 and epithelial differentiation processes .

How does KRT76 expression change during carcinogenesis?

KRT76 is significantly downregulated in human oral squamous cell carcinomas (OSCC), representing the most substantially downregulated structural protein gene in this cancer type . This downregulation strongly correlates with poor prognosis in OSCC patients. In experimental models using the carcinogen 4-nitroquinoline N-oxide (4NQO), KRT76 downregulation is observed as an early event in oral carcinogenesis, first appearing in hyperplastic oral epithelium before progressing through dysplasia to invasive carcinoma .

The pattern of KRT76 loss is often heterogeneous within the same individual, with some lesions showing complete loss while others retain expression, suggesting that multiple mechanisms may regulate KRT76 expression in different tumor microenvironments . When investigating KRT76 expression in tumorigenesis, researchers should examine multiple stages of tumor progression and multiple lesions within the same specimen to account for this heterogeneity.

What are appropriate positive and negative controls when using KRT76 antibodies?

For effective implementation of KRT76 antibodies in research, proper controls are essential:

Positive controls:

• Normal human or mouse differentiated epithelial tissues from the oral cavity, particularly the dorsal tongue and buccal mucosa

• Differentiated layers of the squamous stomach in mice

• Stratified layers of the epidermis

Negative controls:

• KRT76-null mouse tissues (complete absence of staining should be observed)

• Glandular stomach tissue (naturally KRT76-negative)

• Primary antibody omission control

• Non-epithelial tissues such as lymph nodes and thymus, which do not express KRT76

When validating a new KRT76 antibody, researchers should perform simultaneous staining of known positive and negative tissues, ideally including KRT76-knockout tissues if available, to confirm specificity.

How does KRT76 loss impact the immune microenvironment, and how can this be effectively analyzed?

The loss of KRT76 creates profound changes in both local and systemic immune environments. KRT76-deficient mice exhibit splenomegaly and lymphadenopathy with significant increases in regulatory T cells (Tregs), effector T cells, and B cells in secondary lymphoid organs . These changes are accompanied by elevated levels of pro-inflammatory cytokines including IL-6, IL-10, and TNFα in circulation .

To comprehensively analyze these immune changes, researchers should employ:

• Flow cytometric analysis of immune cell populations using markers for:

  • Tregs (TCRβ+CD4+CD3+Foxp3+)

  • Effector T cells (CD4+CD44highCD62Llow)

  • B cells (B220+TCRβ-)

• Cytokine profiling of:

  • Serum for systemic inflammation (IFNγ, IL-2, IL-4, IL-6, IL-10, TNFα)

  • Tissue lysates for local inflammation

  • Interstitial fluid from affected tissues

• Immunohistochemistry to quantify CD45+ infiltrating immune cells in the target tissues

• Analysis of immune cell functional capacity (e.g., suppressive capacity of Tregs or proliferative capacity of effector T cells)

These approaches should be applied both at baseline and after experimental interventions to fully understand the immunomodulatory effects of KRT76 .

What is the relationship between KRT76 expression and regulatory T cell (Treg) functions in the tumor microenvironment?

KRT76 loss creates a complex immunoregulatory environment that impacts tumor development. In KRT76-deficient mice:

• Tregs exhibit enhanced suppressive ability, correlating with increased expression of the ectonucleotidases CD39 and CD73 .

• Effector T cells show reduced proliferative capacity compared to controls.

• Tregs accumulate preferentially in the tumor microenvironment, with significantly increased frequency in KRT76-negative tumors compared to adjacent normal tissue.

• The Treg:Teffector ratio in tumors is significantly altered, creating an immunosuppressive microenvironment favorable to tumor growth.

To investigate this relationship, researchers should:

• Perform dual immunofluorescence staining for Foxp3 (Treg marker) and CD8 (cytotoxic T cell marker) in tumor sections

• Analyze expression of functional markers on tumor-infiltrating Tregs (CTLA-4, PD-1, CD39, CD73)

• Conduct in vitro suppression assays using isolated Tregs from KRT76-positive versus KRT76-negative microenvironments

• Evaluate cytokine profiles within the tumor microenvironment with particular attention to immunosuppressive cytokines like IL-10 and TGF-β

How can researchers accurately quantify KRT76 expression changes across different experimental conditions?

For precise quantification of KRT76 expression across experimental conditions, researchers should implement a multi-modal approach:

RNA level quantification:

• RT-qPCR with validated primer sets specific to KRT76

• RNA-seq for genome-wide expression analysis

• In situ hybridization for spatial resolution of mRNA expression

Protein level quantification:

• Western blotting with appropriate loading controls

• Quantitative immunohistochemistry with digital image analysis

• Flow cytometry for single-cell quantification in dissociated tissues

Data analysis considerations:

• Use multiple reference genes for RT-qPCR normalization

• Implement intensity normalization methods for immunohistochemistry

• Account for tissue heterogeneity by analyzing multiple regions

• Develop a standardized scoring system for KRT76 expression (e.g., percentage of positive cells × staining intensity)

When examining KRT76 downregulation in carcinogenesis, researchers should categorize expression patterns as: complete loss, focal loss, or maintained expression, correlating these patterns with histological features and clinical outcomes .

What are the optimal fixation and antigen retrieval methods for KRT76 immunohistochemistry?

For optimal KRT76 immunodetection in tissue samples, the following protocol is recommended:

Fixation:

• 10% neutral-buffered formalin for 24-48 hours (optimal for preserving tissue architecture while maintaining antigenicity)

• Alternatively, 4% paraformaldehyde for 12-24 hours for immunofluorescence applications

Antigen retrieval methods (in order of effectiveness):

• Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) at 95-98°C for 20 minutes

• HIER using EDTA buffer (pH 9.0) at 95-98°C for 20 minutes

• Enzymatic retrieval using proteinase K (less preferred as it may damage tissue morphology)

Important considerations:

• Overfixation can mask KRT76 epitopes and require more aggressive retrieval

• Fresh frozen sections may provide better antigenicity but poorer morphology

• For dual immunofluorescence with differentiation markers (e.g., Loricrin), optimize antigen retrieval conditions for both antigens

• Include positive controls (normal oral epithelium) with each staining batch to ensure protocol effectiveness

How should researchers design experimental protocols to study KRT76's role in carcinogenesis?

Based on established models investigating KRT76's role in carcinogenesis, researchers should consider the following experimental design:

Chemical carcinogenesis model:

• Use 4-nitroquinoline N-oxide (4NQO) at 100 μg/ml in drinking water for 16 weeks

• Monitor lesion development for a total of 28 weeks

• Include appropriate controls (KRT76+/+, KRT76+/-, and KRT76-/- mice)

• Document lesion appearance, progression, and location systematically

• Collect tissue at multiple timepoints to capture the dynamic changes in KRT76 expression

Analysis parameters:

• Tumor incidence and multiplicity

• Time to tumor onset

• Histopathological grading (hyperplasia, dysplasia, carcinoma)

• KRT76 expression status in lesions

• Immune cell infiltration (CD45+ cells, Tregs, effector T cells)

• Cytokine profiling at baseline and during tumor progression

Experimental timeline:

• Early assessment (2 weeks): cytokine changes, initial immune responses

• Intermediate assessment (6-10 weeks): hyperplasia and dysplasia development

• Late assessment (16-28 weeks): carcinoma development and progression

What approaches are recommended for simultaneously analyzing KRT76 expression and immune cell infiltration?

For comprehensive analysis of the relationship between KRT76 expression and immune responses, researchers should employ:

Multiplexed immunofluorescence techniques:

• Sequential staining with KRT76 antibody and immune cell markers (CD45, CD4, CD8, Foxp3)

• Use of spectrally distinct fluorophores for each marker

• Counterstaining with DAPI for nuclear visualization

• Automated multispectral imaging for quantification

Spatial analysis methods:

• Quantify immune cell density in relation to KRT76-positive versus KRT76-negative regions

• Measure distances between immune cells and KRT76-expressing epithelial cells

• Calculate immune cell clustering in different microenvironmental contexts

Alternative approaches:

• Laser capture microdissection of KRT76-positive and negative regions followed by qPCR analysis of immune markers

• Single-cell RNA sequencing of dissociated tumor samples to correlate KRT76 expression with immune cell transcriptional profiles

• Spatial transcriptomics to preserve tissue context while analyzing gene expression patterns

Quantification standards:

• Number of immune cells per high-power field or per mm² of tissue

• Immune cell density within defined distances from KRT76+ or KRT76- epithelium

• Ratio of different immune cell populations in relation to KRT76 expression status

How can researchers address non-specific binding of KRT76 antibodies?

Non-specific binding is a common challenge when working with keratin antibodies due to structural similarities between family members. To minimize this issue:

Antibody selection and validation:

• Use monoclonal antibodies when possible for greater specificity

• Validate antibodies using KRT76-knockout tissues or cells as negative controls

• Perform Western blotting to confirm detection of a single band at the expected molecular weight (~65 kDa)

Blocking optimization:

• Extend blocking time to 2 hours at room temperature using 5-10% normal serum from the same species as the secondary antibody

• Add 0.1-0.3% Triton X-100 to blocking buffer to reduce hydrophobic interactions

• Consider adding 1% BSA to reduce non-specific protein interactions

Additional troubleshooting measures:

• Titrate primary antibody concentration to determine optimal dilution

• Increase washing steps (5 x 5 minutes) with agitation

• Pre-absorb antibody with recombinant keratin proteins (except KRT76)

• Use alternative detection systems if autofluorescence is an issue in immunofluorescence applications

Cross-reactivity testing:

• Test antibody on tissues known to express other keratins but not KRT76

• Perform peptide competition assays to confirm binding specificity

What are the potential confounding factors when interpreting KRT76 immunostaining in tumor samples?

When analyzing KRT76 expression in tumor samples, researchers should be aware of several potential confounding factors:

Tumor heterogeneity:

• KRT76 expression can vary significantly within the same tumor

• Sample multiple regions of each tumor to account for spatial heterogeneity

• Use whole slide scanning and digital pathology tools for comprehensive assessment

Technical considerations:

• Edge effects (increased non-specific staining at tissue margins)

• Crushing artifacts that may alter epitope accessibility

• Variability in fixation quality across the tissue sample

• Background staining from endogenous peroxidases or biotin

Biological confounders:

• Inflammatory infiltrates potentially masking epithelial staining

• Altered differentiation states affecting normal KRT76 expression patterns

• Secondary loss of KRT76 due to tumor necrosis or hypoxia

• Variations in KRT76 expression due to tumor microenvironment influences rather than intrinsic regulation

Interpretation guidelines:

• Always compare to adjacent normal epithelium as an internal control

• Use a standardized scoring system accounting for both intensity and percentage of positive cells

• Correlate KRT76 staining with differentiation markers to distinguish true loss from altered differentiation

• Consider the context of inflammatory infiltrates when interpreting KRT76 loss patterns

How can researchers distinguish between true KRT76 downregulation and technical staining failures?

Differentiating between genuine KRT76 downregulation and technical artifacts requires systematic controls and validation approaches:

Essential controls:

• Internal positive control: Adjacent normal epithelium within the same section should show expected KRT76 positivity

• External positive control: Known KRT76-positive tissue processed simultaneously

• Negative control: Primary antibody omission on serial sections

• Gradient control: Areas transitioning from normal to tumor tissue should show gradual changes in KRT76 expression

Validation strategies:

• Confirm protein-level findings with mRNA expression analysis (RT-qPCR or in situ hybridization)

• Use multiple antibodies targeting different epitopes of KRT76

• Implement dual staining with differentiation markers to assess whether KRT76 loss correlates with differentiation changes

• Perform serial sectioning to confirm consistent patterns across the tissue

Technical validation measures:

• Standardize all steps of immunohistochemistry protocol

• Process all comparative samples in the same batch

• Use automated staining platforms when possible to reduce technical variability

• Employ digital image analysis for objective quantification

Documentation for publication:

• Include representative images of positive and negative controls

• Show transition zones between positive and negative areas

• Document antibody validation methodology

• Report scoring methods with inter-observer concordance measures

What are promising approaches for targeting the KRT76-immune cell axis in cancer therapy?

Based on the immunomodulatory role of KRT76, several therapeutic strategies warrant investigation:

Immunotherapy enhancement approaches:

• Combination therapies targeting both KRT76 restoration and immune checkpoint inhibition

• Selective depletion of Tregs in KRT76-negative tumor microenvironments

• Cytokine modulation targeting the IL-6/TNFα axis that is dysregulated with KRT76 loss

KRT76 restoration strategies:

• Epigenetic modifiers to reverse potential methylation-based silencing of KRT76

• Small molecules that stabilize existing KRT76 protein

• Gene therapy approaches to reintroduce functional KRT76 in tumor cells

Predictive biomarker development:

• KRT76 status as a stratification factor for immunotherapy response

• Combined assessment of KRT76 expression and Treg infiltration as a prognostic tool

• Liquid biopsy approaches to monitor KRT76 status non-invasively

Experimental approaches to investigate these directions:

• Patient-derived xenograft models with KRT76 manipulation

• Ex vivo tumor slice cultures to test pharmacological interventions

• Single-cell sequencing to identify cell populations most affected by KRT76 restoration

• Clinical sample analysis correlating KRT76 status with immunotherapy outcomes

What methodological advances are needed to better understand KRT76's molecular interactions?

To advance understanding of KRT76's molecular functions beyond its structural role, several methodological approaches should be developed:

Protein interaction studies:

• Proximity labeling techniques (BioID, APEX) to identify KRT76 interaction partners in living cells

• Co-immunoprecipitation coupled with mass spectrometry to identify the KRT76 interactome

• FRET/FLIM analysis to study direct protein interactions in situ

Functional domain mapping:

• Generation of domain-specific KRT76 antibodies

• Creation of domain deletion mutants to identify regions critical for immune regulation

• Site-directed mutagenesis of potential post-translational modification sites

Signaling pathway analysis:

• Phosphoproteomics to identify signaling pathways altered by KRT76 loss

• ChIP-seq to identify potential transcriptional regulatory mechanisms

• Interactome analysis focused on immune signaling molecules

Advanced imaging techniques:

• Super-resolution microscopy to study KRT76 filament organization

• Live cell imaging with fluorescently tagged KRT76 to study dynamics

• Correlative light and electron microscopy to link molecular localization with ultrastructural features

These methodological advances would help bridge the gap between KRT76's known structural functions and its emerging role in immune regulation and carcinogenesis .

How might single-cell technologies advance our understanding of KRT76 in tumor heterogeneity?

Single-cell technologies offer unprecedented opportunities to understand the complex relationship between KRT76 expression, cellular heterogeneity, and the tumor microenvironment:

Single-cell RNA sequencing applications:

• Mapping epithelial cell states along the KRT76 expression continuum

• Identifying transcriptional signatures associated with KRT76 loss

• Characterizing immune cell populations in KRT76-positive versus negative microenvironments

• Discovering potential compensatory mechanisms in KRT76-deficient cells

Spatial transcriptomics approaches:

• Correlating KRT76 expression patterns with spatial organization of immune cells

• Identifying microenvironmental niches that influence KRT76 expression

• Mapping signaling gradients associated with KRT76 expression boundaries

Multi-omics integration:

• Combining single-cell transcriptomics with proteomics and epigenomics

• Integrating spatial and temporal dimensions of KRT76 regulation

• Correlating KRT76 status with metabolic profiles at single-cell resolution

Analytical frameworks:

• Trajectory analysis to map the progression of KRT76 loss during carcinogenesis

• Cell-cell communication analysis to identify paracrine interactions between KRT76-expressing cells and immune populations

• Network analysis to identify hub genes coordinating with KRT76 in epithelial-immune communication

These single-cell approaches would provide a comprehensive understanding of the dynamic role of KRT76 in tumor evolution and immune regulation, potentially identifying new therapeutic targets and biomarkers .