MYH11 Antibody

What is MYH11 and where is it expressed in normal tissues?

MYH11, also known as smooth muscle myosin heavy chain (SMMHC) or Myosin-11, belongs to the myosin heavy chain family and functions as a contractile protein that converts chemical energy into mechanical energy through ATP hydrolysis . It is specifically expressed in smooth muscle cells throughout the body, including blood vessels, gastrointestinal tract, and urinary tract . MYH11 expression begins during embryonic development, with detection possible as early as E9.5 in the dorsal aorta of mice, and continues into adulthood . The protein is predominantly localized in the cytoplasm and cell membrane of smooth muscle cells and is also found in myoepithelial cells of the breast .

What types of MYH11 antibodies are available for research, and what are their characteristics?

Several types of MYH11 antibodies are available for research purposes:

• Rabbit Monoclonal Antibodies: These offer high specificity and sensitivity for human MYH11, such as the MYH11/2303R clone, which is suitable for immunohistochemistry on paraffin-embedded tissues (IHC-P) .

• Recombinant Rabbit Monoclonal Antibodies: For example, the MSVA-375R clone, which is designed for immunohistochemistry applications with a recommended dilution of 1:100-1:200 .

Most commercially available MYH11 antibodies are developed against recombinant full-length protein corresponding to human MYH11 . These antibodies typically show cytoplasmic staining patterns in smooth muscle cells and offer varying sensitivities for detecting MYH11 expression during different developmental stages .

How can I validate the specificity of an MYH11 antibody for my research?

Validating MYH11 antibody specificity requires multiple approaches:

• Positive Control Tissues: Use tissues known to express high levels of MYH11, such as colon, where strong staining should be observed in muscularis mucosa, muscular propria, small and medium-sized vessels, and the fine smooth muscle layer surrounding crypt glands .

• Negative Control Tissues: Verify absence of staining in tissues known not to express MYH11, such as colon epithelial cells .

• Comparative Analysis: Compare staining patterns with genetic reporter models, such as the MYH11 knock-in dual reporter mouse model, which can serve as a gold standard for MYH11 expression patterns .

• Antibody Comparison: Test multiple antibodies from different sources, as some MYH11 antibodies may show varying sensitivities, particularly for developmental studies. Research has shown that some antibodies fail to detect MYH11 expression at early embryonic stages (E9.5) while others can successfully identify these cells .

What are the optimal conditions for immunohistochemical detection of MYH11 in tissue samples?

For optimal immunohistochemical detection of MYH11:

• Tissue Fixation: Standard formalin fixation and paraffin embedding procedures are generally suitable for MYH11 detection.

• Antibody Selection: Use validated antibodies such as rabbit monoclonal anti-MYH11 antibodies that are specifically recommended for IHC-P applications .

• Dilution: Follow manufacturer's recommendations; for example, MSVA-375R is typically used at 1:100-1:200 dilution .

• Controls: Always include positive controls (colon tissue with smooth muscle components) and negative controls (epithelial cells) to validate staining specificity .

• Signal Amplification: Consider using signal amplification methods for detecting low-level expression, particularly in developmental studies where direct immunofluorescence may have limited sensitivity compared to genetic reporter systems .

• Antigen Retrieval: Heat-induced epitope retrieval methods are generally effective for MYH11 detection in fixed tissues.

How do MYH11 expression patterns differ across developmental stages, and what methodological approaches are best for studying these changes?

MYH11 expression shows dynamic patterns across developmental stages:

• Early Development: MYH11 expression begins as early as E9.5 in the dorsal aorta of mice, but detection at this stage can be challenging with conventional immunostaining approaches .

• Mid-gestation: By E10.5-11.5, MYH11 expression becomes more robust in vascular structures, with expression detectable in the umbilical cord and major vessels .

• Late Gestation: At E16.5, strong expression is observed in thoracic aorta, gut, lung, and stomach .

• Postnatal: Expression extends to bladder, gallbladder, blood vessels of brain, and coronary arteries from P0-30 .

For studying developmental expression patterns, genetic reporter models often provide superior sensitivity compared to antibody-based detection. The MYH11 knock-in dual reporter mouse model, which expresses nuclear lacZ or H2B-GFP under control of the endogenous MYH11 promoter, offers a particularly sensitive approach for detecting MYH11 expression during development . This model has revealed that some MYH11 antibodies fail to detect early expression that can be visualized with the reporter .

What are the key considerations when using MYH11 antibodies for co-immunostaining with other markers?

When performing co-immunostaining with MYH11 antibodies:

• Antibody Compatibility: Ensure primary antibodies are raised in different host species to avoid cross-reactivity. If using multiple rabbit antibodies, consider sequential staining protocols with intermediate blocking steps.

• Spectral Separation: Choose fluorophores with minimal spectral overlap to avoid false-positive signals from bleed-through.

• Expression Localization: Remember that MYH11 shows cytoplasmic localization, while many other markers may be nuclear or membrane-associated. This differential localization can be advantageous for clear discrimination between markers.

• Fixation Effects: Some co-markers may require different fixation conditions than optimal for MYH11. Test fixation protocols that preserve epitopes for all target proteins.

• Signal Amplification Balance: When co-staining with markers of different expression levels, balance signal amplification methods to avoid overwhelming weak signals or saturating strong signals.

• Validation: Validate co-staining patterns against genetic reporter models when possible, as demonstrated in studies using the MYH11 knock-in dual reporter mouse model alongside other lineage markers .

How can MYH11 antibodies be used to investigate smooth muscle cell phenotypic switching in disease models?

MYH11 antibodies are valuable tools for studying smooth muscle cell (SMC) phenotypic switching:

• Longitudinal Lineage Tracing: Combine MYH11 antibody staining with genetic lineage tracing using the MYH11 knock-in dual reporter mouse model crossed with inducible Cre lines to distinguish cells that maintain MYH11 expression from those that have downregulated it during phenotypic switching .

• Co-staining Approach: Perform co-immunostaining with MYH11 antibodies and markers of synthetic SMC phenotype (such as osteopontin, vimentin) or proliferation markers (Ki67, PCNA) to identify cells in transition.

• Single-cell Analysis: Combine immunostaining with laser capture microdissection or single-cell isolation techniques to analyze gene expression profiles of individual cells showing variable MYH11 expression.

• Temporal Dynamics: Use the dual-labeling capacity of the MYH11 knock-in reporter mouse, where H2B-GFP marks cells that have expressed MYH11 historically while nuclear lacZ marks cells currently expressing MYH11, to distinguish de novo generated SMCs from those arising from preexisting SMCs .

• Quantitative Analysis: Apply digital image analysis to quantify changes in MYH11 expression intensity across different regions of vascular lesions or other pathological tissues.

What are common sources of false-positive and false-negative results when using MYH11 antibodies, and how can these be mitigated?

Common artifacts and their mitigation strategies include:

False-Positive Results:

• Cross-reactivity with other myosin isoforms: Use highly specific monoclonal antibodies validated against tissues known to express different myosin heavy chain isoforms.

• Non-specific binding: Include appropriate blocking steps (serum matching secondary antibody species) and validate with genetic knockout or negative control tissues.

• Autofluorescence: Include unstained control sections to identify autofluorescence patterns, particularly in tissues rich in elastin and collagen like blood vessels.

False-Negative Results:

• Epitope masking: Optimize antigen retrieval methods; different MYH11 antibodies may require specific retrieval conditions.

• Developmental stage sensitivity: Some antibodies fail to detect MYH11 at early developmental stages despite its presence. Compare multiple antibodies or use genetic reporter systems for developmental studies .

• Low expression detection: Consider signal amplification methods or use genetic reporter models which have shown higher sensitivity than direct immunostaining for detecting low levels of MYH11 expression .

How can MYH11 antibodies be used in combination with lineage tracing to study smooth muscle cell origins and heterogeneity?

MYH11 antibodies, particularly when used alongside genetic lineage tracing tools, enable sophisticated analysis of SMC origins and heterogeneity:

• Dual Reporter Systems: The MYH11 knock-in dual reporter mouse model can be crossed with tissue-specific Cre lines (such as Wnt1-Cre for neural crest or Mef2c-Cre for second heart field) to trace the developmental origins of SMCs in different vascular beds .

• Clonal Analysis: Induce sparse labeling with low doses of tamoxifen in inducible Cre systems crossed with the MYH11 reporter line to perform clonal analysis of labeled H2B-GFP+ cells, revealing mechanisms of vascular SMC organization .

• Marker Co-expression: Combine MYH11 antibody staining with other lineage-specific markers to identify heterogeneous populations within the smooth muscle compartment.

• Temporal Induction: Use temporally controlled Cre activation at different developmental stages to determine when specific SMC populations arise and how they contribute to vessel formation.

• Disease Modeling: Apply these techniques in disease models to understand how different SMC populations respond to pathological stimuli, potentially revealing population-specific therapeutic targets.

How are MYH11 mutations associated with thoracic aortic aneurysms, and what research applications do MYH11 antibodies have in this context?

MYH11 mutations have been identified in approximately 1% of patients with familial predisposition to thoracic aortic aneurysms leading to acute aortic dissections (TAAD) . These mutations typically affect the contractile function of smooth muscle cells, compromising vascular wall integrity. Research applications of MYH11 antibodies in this context include:

• Mutation Impact Assessment: Compare MYH11 protein localization and expression levels in tissues from patients with different MYH11 mutations versus controls to establish genotype-phenotype correlations.

• Altered Contractility Visualization: Combine MYH11 antibody staining with markers of contractile apparatus organization to assess how mutations affect the structural integrity of the smooth muscle contractile unit.

• Mechanistic Studies: Use MYH11 antibodies in cellular and tissue models carrying disease-associated mutations to investigate downstream effects on mechanotransduction, cell adhesion, and ECM interactions.

• Biomarker Development: Assess whether altered MYH11 expression patterns in circulating cells or accessible tissues might serve as biomarkers for disease progression or therapeutic response.

• Therapeutic Target Validation: Apply MYH11 antibodies to validate targets in signaling pathways affected by MYH11 mutations, which might represent intervention points for novel therapeutics.

What is the significance of MYH11 gene rearrangements in acute myeloid leukemia, and how can antibodies aid in studying these mechanisms?

The MYH11 gene is involved in genomic rearrangements inv(16)(p13q22) and t(16;16)(p13;q22), which are characteristic of acute myeloid leukemia with abnormal bone marrow eosinophils (AML-M4Eo) . These rearrangements result in a fusion protein composed of core binding factor beta (CBF-beta) with SMMHC (CBFbeta-SMMHC), which localizes to the nuclei of leukemic cells . MYH11 antibodies can aid in studying these mechanisms through:

• Fusion Protein Detection: Specialized antibodies that recognize epitopes retained in the fusion protein can be used to detect and localize CBFbeta-SMMHC in patient samples and experimental models.

• Diagnostic Applications: Immunohistochemical detection of the fusion protein may complement cytogenetic and molecular testing in diagnostically challenging cases.

• Mechanistic Studies: Combining MYH11 antibodies with chromatin immunoprecipitation (ChIP) can identify genomic targets of the fusion protein, elucidating its role in leukemogenesis.

• Therapeutic Response Monitoring: MYH11 antibodies can be used to monitor changes in fusion protein expression or localization following experimental therapeutics targeting this oncogenic mechanism.

• Minimal Residual Disease: Sensitive detection methods using MYH11 antibodies might contribute to monitoring minimal residual disease in patients with inv(16) AML.

How does the MYH11 knock-in dual reporter mouse model advance smooth muscle cell research beyond traditional antibody-based methods?

The MYH11 knock-in dual reporter mouse model represents a significant advancement in SMC research by offering several advantages over traditional antibody-based methods:

• Enhanced Sensitivity: The nuclear localization of reporter proteins (H2B-GFP and nuclear lacZ) concentrates the signal in a small nuclear volume, dramatically improving detection sensitivity compared to cytoplasmic staining with antibodies, particularly at early developmental stages (as early as E9.5) .

• Dual Labeling Capability: The model contains a LoxP-nlacZ-4XpolyA-LoxP-H2B-GFP-polyA reporter cassette that allows for conditional switching from nuclear lacZ to H2B-GFP expression upon Cre-mediated recombination, enabling sophisticated lineage tracing experiments .

• Live Imaging Potential: H2B-GFP expression allows for live visualization of MYH11-expressing cells, enabling dynamic studies not possible with antibody staining of fixed tissues .

• Faithful Expression Pattern: The reporter genes are knocked into the endogenous MYH11 locus, ensuring that their expression accurately reflects endogenous MYH11 expression patterns without the positional effects common in transgenic models .

• Broad Applicability: Unlike some transgenic models (such as the Myh11-CreERT2 transgenic line inserted on the Y chromosome), the knock-in model can be used in both male and female mice, broadening its experimental utility .

• Clonal Analysis Capability: When crossed with inducible Cre lines and induced with low doses of tamoxifen, the model allows for clonal analysis of SMC organization and development .

What advanced experimental approaches combine MYH11 antibodies with other molecular techniques for comprehensive smooth muscle cell characterization?

Cutting-edge research approaches that integrate MYH11 antibody detection with other molecular techniques include:

• Single-Cell Multi-omics: Combining immunostaining for MYH11 with single-cell RNA sequencing, ATAC-seq, or proteomics to correlate protein expression with transcriptomic, epigenetic, or proteomic profiles at single-cell resolution.

• Spatial Transcriptomics: Integrating MYH11 immunofluorescence with spatial transcriptomics techniques to map gene expression patterns in the context of tissue architecture while confirming smooth muscle cell identity.

• CRISPR Screening: Using MYH11 expression as a phenotypic readout in CRISPR-based functional genomics screens to identify regulators of smooth muscle cell differentiation or phenotypic switching.

• Intravital Imaging: Combining MYH11 reporter models with intravital microscopy techniques to observe smooth muscle cell behavior in living organisms during development or in response to disease stimuli.

• Organoid Models: Applying MYH11 antibodies to identify and characterize smooth muscle cells in organoid cultures, potentially using sorting strategies based on reporter expression to enrich for specific cell populations.

• Computational Modeling: Integrating quantitative MYH11 expression data from antibody-based imaging with computational models of vascular mechanics to predict how changes in contractile protein expression affect vascular function.