KRT76 Antibody, FITC conjugated

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

KRT76 (Keratin 76) is a cytoskeletal intermediate filament protein that belongs to the keratin family. It is specifically expressed in the differentiated epithelial layers of the skin, oral cavity, and squamous stomach . Additional expression has been detected in the palatal and gingival epithelium, vagina, and eyelid . KRT76 is considered a structural marker protein with tissue-specific expression, though emerging research suggests it plays roles beyond structural integrity . Its molecular weight ranges between 40-67kDa, typical of keratin family proteins .

What is the functional significance of KRT76 in epithelial tissues?

KRT76 serves critical functions in maintaining epithelial tissue integrity and barrier function. Studies using KRT76 knockout mice have demonstrated that this protein is essential for:

• Maintenance of proper tight junction function through interaction with Claudin-1 (CLDN1)

• Regulation of skin homeostasis and wound healing

• Postnatal survival (knockout mice die before 12 weeks of age)

• Immune system modulation and prevention of excessive inflammation

Loss of KRT76 leads to neonatal skin flaking, hyperpigmentation, inflammation, and impaired wound healing, highlighting its essential role in epithelial barrier function .

How is KRT76 expression altered in pathological conditions?

KRT76 expression is frequently downregulated in several disease states:

• In oral squamous cell carcinomas (OSCC), reduced KRT76 expression correlates with poor prognosis

• Downregulation is observed during oral carcinogenesis in both human samples and in hamster models of oral cancer

• Loss of KRT76 is associated with increased inflammation and immune dysregulation

Table 1: KRT76 Expression Patterns in Normal vs. Pathological Conditions

What experimental models exist for studying KRT76 function?

Several experimental models have been developed to study KRT76 function:

• Genetic knockout models: KRT76 knockout mice (KRT76 KO or KRT76^-/-) exhibit phenotypes including skin flaking, hyperpigmentation, inflammation, impaired wound healing, and premature death . These mice show spleen and lymph node enlargement along with increased regulatory T cells and inflammatory cytokines .

• Conditional knockout models: Inducible systems allow for temporal control of KRT76 deletion, revealing hyperproliferation and wound-related expansion of the interfollicular epidermis .

• Chemical carcinogenesis models: DMBA treatment of hamster buccal pouch has been used to study KRT76 expression changes during oral carcinogenesis .

These models provide valuable platforms for investigating the roles of KRT76 in tissue homeostasis, immune regulation, and cancer progression.

How does KRT76 interact with tight junction proteins?

KRT76 plays a crucial role in tight junction functionality through direct interaction with CLDN1 (Claudin-1), an integral tight junction protein. Research findings indicate:

• KRT76 is required for proper localization of CLDN1 in tight junctions

• In KRT76 knockout mice, CLDN1 becomes mislocalized, leading to functionally defective tight junctions

• This interaction establishes a previously unrecognized connection between the intermediate filament cytoskeleton network and tight junctions

• The mislocalization of CLDN1 has been linked to various skin diseases, including psoriasis

This mechanism reveals a novel regulatory function of KRT76 beyond its structural role, highlighting the importance of cytoskeletal components in maintaining epithelial barrier integrity.

What is the relationship between KRT76 and immune regulation?

Recent research has uncovered an unexpected immunomodulatory role for KRT76:

• KRT76 knockout mice exhibit spleen and lymph node enlargement

• Loss of KRT76 leads to increased effector T cells and regulatory T cells (Tregs)

• Pro-inflammatory cytokines are elevated in KRT76-deficient mice

• Local inflammation is observed in epithelia normally expressing KRT76 (tongue, squamous stomach, skin) with significant increases in immune cell infiltrate (CD45+ cells)

• Despite inflammatory changes, barrier function as assessed by dye penetration assays remains intact in oral epithelia of knockout mice

These findings suggest KRT76 functions beyond structural support, potentially influencing immune cell activation and cytokine production in epithelial tissues.

What are the optimal conditions for using KRT76 antibody in immunofluorescence?

For optimal immunofluorescence staining with KRT76 antibody (FITC conjugated), the following protocol is recommended based on published methodologies:

• Tissue preparation: Fix tissues in cold methanol for 10 minutes

• Deparaffinization and antigen retrieval: For formalin-fixed paraffin-embedded tissues, standard deparaffinization followed by antigen retrieval is essential

• Blocking: Block with 5% normal goat serum containing 0.3% (v/v) Triton X-100 in PBS for 1 hour at room temperature

• Primary antibody: Incubate with non-conjugated KRT76 antibody at 1:250 dilution overnight at 4°C, or use directly conjugated FITC-KRT76 antibody according to manufacturer recommendations

• Secondary antibody: If using unconjugated primary, incubate with Alexa Fluor 488 anti-rabbit antibody at 1:200 dilution for 1 hour at room temperature

• Nuclear counterstain: Counterstain with DAPI and mount appropriately for fluorescence microscopy

For FITC-conjugated antibodies specifically, minimize exposure to light throughout the procedure to prevent photobleaching.

How should KRT76 antibody be optimized for flow cytometry?

When using KRT76 antibody (FITC conjugated) for flow cytometry, consider these optimization steps:

• Concentration determination: Typically 0.5-1 μg per million cells is recommended as a starting point

• Sample preparation: Proper fixation (typically with 2-4% paraformaldehyde) followed by permeabilization is required for intracellular keratins

• Controls: Always include:

  • Unstained cells

  • Isotype control (FITC-conjugated IgG of matching isotype)

  • Single-color controls for compensation when performing multicolor analysis

  • Positive control (e.g., epithelial cell line known to express KRT76)

Table 2: Troubleshooting KRT76-FITC Flow Cytometry Analysis

What validation steps should be performed for KRT76 antibody specificity?

Validating antibody specificity is crucial for obtaining reliable research data. For KRT76 antibody, consider these validation approaches:

• Positive and negative control tissues: Test the antibody on tissues known to express or lack KRT76 (positive: oral epithelium, squamous stomach; negative: most non-epithelial tissues)

• Knockout validation: Compare staining between wild-type and KRT76 knockout tissues (if available)

• Peptide competition assay: Pre-incubate antibody with immunizing peptide to demonstrate specificity

• Multiple antibody comparison: Use alternative KRT76 antibodies targeting different epitopes to confirm staining patterns

• Western blot correlation: Confirm that immunofluorescence/IHC staining correlates with western blot results in the same samples

• RNA expression correlation: Compare protein detection with mRNA expression data (e.g., qRT-PCR) from the same samples

These validation steps help ensure that observed signals truly represent KRT76 rather than non-specific staining or cross-reactivity with other keratins.

What are recommended protocols for immunohistochemical detection of KRT76?

For optimal immunohistochemical detection of KRT76 in formalin-fixed tissues:

• Antigen retrieval: Boil tissue sections in 10 mM citrate buffer (pH 6.0) for 10-20 minutes followed by cooling at room temperature for 20 minutes

• Blocking: For human tissues, block with 5% normal goat serum containing 0.3% Triton X-100; for animal models, use 3% BSA and 2% goat serum

• Primary antibody: Apply KRT76 antibody at 0.25-0.5 μg/mL concentration for 30 minutes at room temperature

• Secondary antibody: Use appropriate biotin-conjugated secondary antibody (e.g., biotin-conjugated anti-rabbit raised in goat)

• Detection: Develop signal using standard ABC-DAB procedure or other appropriate detection system

• Controls: Include positive control tissues (skin, adeno- or squamous carcinomas)

This protocol can be modified depending on the specific tissue type and experimental requirements.

How can KRT76 antibodies be applied to study cancer progression?

KRT76 antibodies are valuable tools for studying cancer progression, particularly in epithelial malignancies:

• Diagnostic applications: KRT76 downregulation correlates with poor prognosis in oral squamous cell carcinomas, making it a potential prognostic marker

• Cancer progression studies: Monitoring KRT76 expression changes during carcinogenesis can provide insights into disease mechanisms

• Experimental approaches:

  • Immunohistochemistry of tumor biopsies to assess KRT76 expression levels

  • Flow cytometry to quantify KRT76 expression in isolated tumor cells

  • Co-staining with proliferation markers to correlate KRT76 loss with increased proliferation

  • Analysis of KRT76 in relation to markers of epithelial-mesenchymal transition

• Animal models: Using KRT76 antibodies to track expression changes in experimental carcinogenesis models such as the DMBA-treated hamster buccal pouch

What are the considerations for multiplexed analysis with KRT76-FITC antibodies?

When performing multiplexed analysis with KRT76-FITC antibodies:

• Spectral compatibility: FITC (emission peak ~520 nm) should be paired with fluorophores having minimal spectral overlap (e.g., PE, APC)

• Sequential staining: For complex protocols, consider sequential rather than simultaneous antibody incubations

• Panel design for flow cytometry:

  • Include markers for cell identification (e.g., epithelial markers)

  • Add functional markers relevant to your research question

  • Ensure appropriate compensation controls for each fluorophore

• Microscopy considerations:

  • For co-localization studies with tight junction proteins, include markers like CLDN1

  • For inflammation studies, consider co-staining with immune cell markers (CD45, etc.)

  • Use appropriate filter sets to minimize bleed-through between channels

Careful optimization of staining protocols and imaging/acquisition parameters is essential for obtaining reliable multiplexed data.

How can weak or non-specific KRT76 staining be addressed?

When encountering weak or non-specific staining with KRT76 antibodies:

• Weak signal solutions:

  • Optimize antibody concentration (try range from 0.25-2 μg/mL)

  • Enhance antigen retrieval (extend boiling time in citrate buffer)

  • Increase primary antibody incubation time (overnight at 4°C)

  • Use signal amplification systems (e.g., tyramide signal amplification)

• Non-specific binding solutions:

  • Increase blocking time/concentration

  • Use additional blocking agents (e.g., add 0.05% BSA to blocking buffer)

  • Include detergents (0.05-0.3% Triton X-100) to reduce background

  • Pre-absorb antibody with non-specific proteins

• Tissue-specific considerations:

  • For skin samples: Address potential autofluorescence with Sudan Black treatment

  • For oral tissues: Optimize fixation time to prevent overfixation

  • For gastric tissues: Consider specialized fixatives for optimal epitope preservation

Table 3: KRT76 Staining Optimization by Application

What emerging applications exist for studying KRT76 in disease models?

Recent discoveries about KRT76 function suggest several promising research directions:

• Tight junction regulation: Further investigation into how KRT76 regulates CLDN1 localization and tight junction assembly could provide insights into epithelial barrier disorders

• Immune modulation: Exploring the mechanistic basis for KRT76's role in regulating inflammatory responses and T cell populations could lead to novel immunotherapy approaches

• Cancer progression models: Using KRT76 as a marker in longitudinal studies of carcinogenesis to identify critical transition points

• Therapeutic targeting: Developing strategies to maintain or restore KRT76 expression in cancers where its downregulation correlates with poor outcomes

• Wound healing applications: Investigating KRT76's role in wound repair mechanisms could lead to new approaches for treating chronic wounds

These emerging areas highlight the evolving understanding of KRT76 beyond its classical structural role, positioning it as a multifunctional protein with significant implications for epithelial health and disease.