MYH11 Antibody, FITC conjugated

What is MYH11 and why is it an important research target?

MYH11 encodes smooth muscle myosin heavy chain (SMMHC), a major contractile protein produced in smooth muscle tissues. It plays a crucial role in muscle contraction and cellular movement by interacting with actin filaments to generate force . This interaction is vital for various physiological processes, including vasoconstriction and gastrointestinal motility, making MYH11 an important target for studying smooth muscle function and related disorders .

At the molecular level, MYH11 exists as part of a hexameric assembly, which includes two heavy chain subunits and light chain subunits that can be either phosphorylatable or non-phosphorylatable . The phosphorylation state of the myosin light chain serves as a key regulatory mechanism for smooth muscle contraction, modulated by calcium/calmodulin-dependent myosin light chain kinase .

What applications are FITC-conjugated MYH11 antibodies suitable for?

FITC-conjugated MYH11 antibodies are specifically designed for fluorescence-based applications. Based on the available research data, these antibodies are suitable for:

• Immunofluorescence (IF) – Particularly useful for tissue localization studies with recommended dilutions of 1:50-200 (based on 0.5 mg/ml concentration)

• Flow cytometry (FCM) – For analyzing MYH11 expression in cell populations

• Immunohistochemistry on paraffin-embedded tissues (IHC-P) – When combined with appropriate antigen retrieval methods

The FITC (fluorescein isothiocyanate) conjugation eliminates the need for secondary antibody incubation, reducing background and cross-reactivity issues in multi-color immunofluorescence experiments.

What is the species reactivity profile of available MYH11 antibodies?

Available MYH11 antibodies show different species reactivity profiles depending on the manufacturer and clone:

When selecting an antibody for cross-species studies, researchers should consider antibodies with broader reactivity profiles such as E4N8M or G-4 clones .

What are the recommended storage conditions for FITC-conjugated antibodies?

FITC-conjugated antibodies, including MYH11 antibodies, require specific storage conditions to maintain fluorophore integrity and antibody functionality:

• Temperature: Store at 2-8°C for short-term (1-2 weeks) and at -20°C for long-term storage

• Protection from light: FITC is sensitive to photobleaching, so vials should be wrapped in aluminum foil or stored in opaque containers

• Avoid freeze-thaw cycles: Repeated freezing and thawing can damage both the antibody and the fluorophore

• Do not aliquot certain antibodies: Some manufacturers specifically note "Do not aliquot the antibody"

Following these storage recommendations will help preserve the signal intensity and specificity of FITC-conjugated MYH11 antibodies during extended research projects.

How can FITC-conjugated MYH11 antibodies be optimized for immunofluorescence applications?

Optimizing immunofluorescence protocols for FITC-conjugated MYH11 antibodies requires attention to several technical parameters:

Fixation method: For smooth muscle tissues, 4% paraformaldehyde is generally preferred over alcoholic fixatives to preserve MYH11 antigenicity.

Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) is recommended for formalin-fixed, paraffin-embedded tissues.

Blocking: Use 5-10% normal serum from the same species as the secondary antibody (if a secondary amplification step is needed).

Dilution optimization: Starting with the manufacturer's recommended dilution (e.g., 1:50-200 for polyclonal antibodies ), perform a dilution series to determine optimal signal-to-noise ratio for your specific tissue or cell type.

Counterstaining considerations: When performing nuclear counterstaining, avoid propidium iodide which has spectral overlap with FITC; DAPI or Hoechst are preferred alternatives.

Mounting medium: Use an anti-fade mounting medium specifically formulated for fluorescence preservation with FITC fluorophores.

Validation studies show that careful optimization of these parameters produces consistent staining of smooth muscle structures in various tissues, as demonstrated in immunohistochemical analyses of breast carcinoma, leiomyosarcoma, and colon carcinoma tissues .

How can MYH11 antibodies be used to investigate aortic diseases?

MYH11 antibodies serve as valuable tools for investigating aortic diseases, particularly those involving smooth muscle cell dysfunction:

• Tissue analysis: MYH11 antibodies can quantify smooth muscle content in aortic tissues, which is particularly relevant since mutations in MYH11 cause medial degeneration of the aorta with very low smooth muscle cell content .

• Disease phenotyping: Immunostaining for MYH11 helps characterize disease symptoms including aortic stiffness, aneurysm/dissection, and ductus arteriosus .

• Mutation-phenotype correlation studies: Using these antibodies in conjunction with genetic analysis allows researchers to correlate specific MYH11 mutations with patterns of smooth muscle dysfunction.

Methodology:

• For paraffin-embedded aortic tissue sections, use 1:100-1:400 dilution of primary antibody

• Include both normal and pathological samples for comparative analysis

• Consider dual staining with proliferation or apoptosis markers to assess smooth muscle cell dynamics in disease progression

This approach has yielded insights into the pathogenesis of aortic diseases characterized by smooth muscle cell dysfunction and the molecular mechanisms underlying aortic wall integrity.

What controls should be included when using FITC-conjugated MYH11 antibodies?

Rigorous experimental design requires appropriate controls when using FITC-conjugated MYH11 antibodies:

Positive controls:

• Tissues with known high MYH11 expression (e.g., vascular smooth muscle, myometrium, intestinal smooth muscle)

• Cell lines with confirmed MYH11 expression (e.g., primary smooth muscle cells)

Negative controls:

• Isotype control: FITC-conjugated non-specific IgG from the same host species (rabbit or mouse depending on the antibody)

• Absorption control: Pre-incubation of the antibody with excess MYH11 recombinant protein

• Tissues known to lack MYH11 expression (e.g., skeletal muscle, cardiac muscle)

Technical controls:

• Autofluorescence control: Unstained tissue section to account for natural tissue fluorescence

• Single-color controls: Essential for spectral compensation when performing multicolor immunofluorescence

• Secondary-only control (if using a detection system): Omit primary antibody to assess non-specific binding

Including these controls allows researchers to confidently interpret staining patterns and distinguish specific MYH11 signals from background or artifact.

How can MYH11 antibodies contribute to leukemia research?

MYH11 antibodies have specific applications in leukemia research, particularly in studying the CBFB-MYH11 fusion associated with acute myeloid leukemia (AML):

• Detection of fusion proteins: MYH11 antibodies can detect the chimeric CBFB-MYH11 protein that results from one of the most frequent chromosomal translocations associated with AML .

• Mechanistic studies: These antibodies help investigate how the fusion protein suppresses the transactivation mediated by CBF/AML-1 and impairs normal hematopoietic differentiation .

• Identification of preleukemic progenitors: MYH11 antibodies aid in identifying preleukemic myeloid progenitors that can be targets for AML transformation .

Experimental approach:

• Use flow cytometry with FITC-conjugated MYH11 antibodies to identify cells expressing the fusion protein

• Combine with markers of hematopoietic differentiation for comprehensive phenotyping

• Consider protein-protein interaction studies to investigate how the fusion protein interacts with transcriptional machinery

This research direction has significant implications for understanding leukemogenesis and developing targeted therapies for AML patients with CBFB-MYH11 fusions.

What are the technical considerations for using FITC-conjugated MYH11 antibodies in flow cytometry?

Flow cytometric analysis with FITC-conjugated MYH11 antibodies requires attention to several technical aspects:

Sample preparation:

• For intracellular staining, use a fixation/permeabilization buffer that preserves MYH11 epitopes

• Optimize cell concentration to 1-5 × 10^6 cells/mL for consistent results

• Ensure single-cell suspensions by filtering through a 40-70 μm cell strainer

Antibody titration:

• Determine optimal antibody concentration using a serial dilution experiment

• Plot the signal-to-noise ratio versus antibody concentration to identify the optimal dilution point

Instrument setup:

• FITC is excited by the 488 nm laser and detected in the 530/30 nm channel

• Perform proper compensation if using multiple fluorophores, especially those with spectral overlap with FITC (e.g., PE, GFP)

Analysis considerations:

• Use appropriate gating strategies to exclude dead cells and debris

• When analyzing tissues with variable MYH11 expression, use histogram overlays rather than percentage positive cells

• Consider using median fluorescence intensity (MFI) for quantitative comparisons

These technical considerations help ensure reliable and reproducible flow cytometry results when studying MYH11 expression in various cell populations.

What are common troubleshooting strategies for weak or absent MYH11 staining?

When encountering weak or absent MYH11 staining, consider the following troubleshooting approaches:

• Antigen retrieval optimization:

  • Test multiple antigen retrieval methods (heat-induced vs. enzymatic)

  • Extend antigen retrieval time for highly fixed tissues

  • Adjust pH of retrieval buffer (try both acidic and basic conditions)

• Antibody concentration adjustment:

  • Increase antibody concentration if signal is weak

  • For paraffin sections, try a higher concentration (1:100) than recommended for frozen sections

• Signal amplification methods:

  • Consider using a biotin-streptavidin amplification system

  • Employ tyramide signal amplification (TSA) for very low abundance targets

  • Use a brighter secondary antibody with multiple fluorophores per molecule

• Fixation considerations:

  • Excessive fixation can mask epitopes; reduce fixation time in future experiments

  • For archived tissues, extend antigen retrieval time

  • Try different fixatives for prospective studies

• Storage-related issues:

  • FITC can photobleach during storage; ensure antibodies are protected from light

  • Check antibody expiration date and storage conditions

  • Avoid repeated freeze-thaw cycles

Methodical testing of these variables while maintaining appropriate controls will help identify the source of staining problems.

How can multiplexed immunofluorescence be achieved with FITC-conjugated MYH11 antibodies?

Multiplexed immunofluorescence allows simultaneous detection of multiple antigens alongside MYH11. To successfully implement this approach:

Panel design considerations:

• Select fluorophores with minimal spectral overlap with FITC (Ex: 490nm, Em: 525nm)

• Compatible fluorophores include Cy3, Cy5, Texas Red, and Alexa Fluor 647

• Plan the staining sequence based on antibody species to avoid cross-reactivity

Sequential staining protocol:

• Perform antigen retrieval as appropriate for all target antigens

• Block with serum or commercially available blocking buffer

• Apply FITC-conjugated MYH11 antibody (1:50-200 dilution)

• Apply additional primary antibodies (from different host species than MYH11)

• Apply species-specific secondary antibodies for non-conjugated primaries

• Counterstain nuclei with DAPI

• Mount with anti-fade mounting medium

Technical considerations:

• Perform spectral unmixing if using fluorophores with overlapping emission spectra

• Include single-stained controls for each fluorophore

• Consider tyramide signal amplification for low-abundance targets

• Use automated multispectral imaging platforms for consistent results

This approach allows researchers to examine the relationship between MYH11 expression and other proteins of interest in the context of tissue architecture.

How does the choice between monoclonal and polyclonal MYH11 antibodies affect research outcomes?

The search results reveal both monoclonal and polyclonal MYH11 antibodies are available, each with distinct advantages:

Selection guidance:

• For detecting MYH11 in fixed tissues where antigen may be partially denatured, polyclonal antibodies often provide superior sensitivity

• For applications requiring precise epitope recognition (e.g., distinguishing specific isoforms), monoclonal antibodies are preferred

• When working with samples from multiple species, select antibodies validated across your species of interest

• For quantitative studies, recombinant monoclonal antibodies offer superior lot-to-lot consistency

Understanding these differences allows researchers to select the most appropriate antibody type for their specific experimental questions and conditions.

How can FITC-conjugated MYH11 antibodies be used in vascular disease research?

FITC-conjugated MYH11 antibodies provide valuable tools for investigating vascular pathologies through several experimental approaches:

• Phenotypic characterization of smooth muscle cells:

  • Distinguish contractile from synthetic phenotypes in vascular disease models

  • Quantify MYH11 expression changes during phenotypic switching

  • Correlate with expression of other contractile proteins

• Lineage tracing in vascular remodeling:

  • Track smooth muscle cell migration in atherosclerotic plaques

  • Identify the origin of intimal cells after vascular injury

  • Study smooth muscle cell contribution to neointima formation

• Evaluation of therapeutic interventions:

  • Assess recovery of contractile phenotype after drug treatment

  • Quantify changes in smooth muscle content after vascular interventions

  • Monitor tissue-engineered vascular graft maturation

Methodological approach:

• Use flow cytometry with FITC-conjugated MYH11 antibodies to quantify expression levels in cell suspensions

• Apply immunofluorescence imaging to preserve spatial relationships within the vessel wall

• Consider co-staining with proliferation markers (Ki-67) or synthetic phenotype markers (osteopontin)

This research direction has implications for understanding and treating conditions like atherosclerosis, aneurysms, and restenosis after vascular interventions.

What are the considerations for using MYH11 antibodies in cancer research beyond leukemia?

MYH11 antibodies have important applications in solid tumor research, particularly for tumors with smooth muscle components or origin:

• Diagnostic applications:

  • Differentiate smooth muscle tumors (leiomyomas, leiomyosarcomas) from other mesenchymal neoplasms

  • Identify smooth muscle differentiation in mixed tumors

  • Characterize the vascular component of tumors

• Tumor microenvironment studies:

  • Investigate pericyte coverage of tumor vasculature

  • Study tumor-associated vasculature maturation

  • Examine the role of smooth muscle cells in metastatic niches

• Epithelial-mesenchymal transition research:

  • Track acquisition of smooth muscle markers in cancer progression

  • Investigate correlations between MYH11 expression and invasion/metastasis

Technical guidance:

• For diagnostic IHC, use optimized protocols with 1:100-1:400 dilution for paraffin sections

• Include appropriate controls, particularly other smooth muscle markers (α-SMA, calponin)

• Confirm specificity using tissues with known MYH11 expression patterns

The search results specifically mention immunohistochemical analyses of MYH11 in breast carcinoma, leiomyosarcoma, colon carcinoma, and angiosarcoma tissues, highlighting its utility in cancer research .

How can phosphorylation state of myosin be studied in relation to MYH11 function?

Studying myosin phosphorylation state in relation to MYH11 function is crucial for understanding smooth muscle contractile mechanisms:

• Methodological approaches:

  • Use phospho-specific antibodies against myosin light chain in combination with MYH11 antibodies

  • Apply proximity ligation assays to detect associations between MYH11 and phosphorylated regulatory proteins

  • Implement FRET-based biosensors to monitor real-time phosphorylation in living cells

• Experimental design for phosphorylation studies:

  • Compare basal versus stimulated conditions (e.g., before and after calcium ionophore treatment)

  • Include phosphatase inhibitors in all sample preparation steps

  • Consider time-course experiments to capture phosphorylation dynamics

• Technical considerations:

  • The phosphorylation state of myosin light chain is a key regulatory mechanism for smooth muscle contraction

  • This regulation is modulated by calcium/calmodulin-dependent myosin light chain kinase

  • Different fixation methods may preserve phospho-epitopes to varying degrees

This research direction provides mechanistic insights into smooth muscle physiology and pathophysiology, helping to elucidate the molecular basis of contractile dysfunction in various disease states.

What are the latest developments in MYH11 antibody technology?

Recent advances in MYH11 antibody technology have expanded research capabilities:

• Recombinant antibody production:

  • Newer antibodies like MYH11 (E4N8M) and MYH11/7610R are produced recombinantly, offering superior lot-to-lot consistency, continuous supply, and animal-free manufacturing

  • These technologies address reproducibility concerns in antibody-based research

• Expanded conjugate options:

  • Beyond FITC, MYH11 antibodies are now available with various conjugates including:

    • Horseradish peroxidase (HRP)

    • Phycoerythrin (PE)

    • Multiple Alexa Fluor® conjugates

  • This variety enables more flexible experimental design and multiplexed analyses

• Application-optimized formulations:

  • Specialized antibody formulations for specific applications (e.g., IHC-P, IF, FCM, ELISA)

  • Carrier-free options for custom conjugation or specialized applications