MYH4 Antibody, FITC conjugated

What is MYH4 and why is it significant in skeletal muscle research?

MYH4 (Myosin Heavy Chain 4) encodes a skeletal muscle protein specifically associated with fast-glycolytic type IIb myofibers. The significance of MYH4 in research stems from its unique expression pattern across species. Notably, MYH4 is typically not expressed at mRNA or protein levels in human skeletal muscle under normal physiological conditions, despite being readily detectable in rodent models . Recent breakthrough research has demonstrated that MYH4 expression can be robustly induced in human skeletal muscle cells through overexpression of large MAF transcription factors (MAFA, MAFB, and MAF), increasing expression by 100-1000 fold compared to controls . This finding indicates that while the genetic program for MYH4 expression remains intact in humans, it is evolutionarily silenced but can be experimentally reactivated, opening new avenues for muscle biology research.

What detection methods are available for MYH4 in experimental systems?

Multiple detection approaches exist for MYH4 identification across different experimental systems. For protein detection, researchers can employ western blotting using specific anti-MYH4 antibodies, which typically detect a band at approximately 223-230 kDa in skeletal muscle lysates . Immunohistochemistry and immunofluorescence methods allow visualization of MYH4 in tissue sections, with FITC-conjugated antibodies providing direct fluorescent detection capabilities . For mRNA detection, quantitative PCR methods have successfully quantified MYH4 transcript levels, particularly in experimental systems with MAF-induced expression . More sensitive detection of low-abundance MYH4 transcripts can be achieved through RNAscope fluorescent in situ hybridization, which has successfully visualized MYH4 RNA puncta in human myotubes overexpressing large MAF transcription factors . For definitive protein identification, LC-MS/MS analysis has confirmed the presence of MYH4-specific peptides in human myotubes experimentally manipulated to express MYH4 .

How does MYH4 expression differ between human and rodent skeletal muscle?

MYH4 expression exhibits significant interspecies variation that researchers must consider when designing experiments and interpreting results. In rodent models (mice and rats), MYH4 is abundantly expressed in skeletal muscles, particularly those with predominant fast-glycolytic fiber composition, and its protein product is readily detectable using standard antibody-based methods . In contrast, human skeletal muscle typically lacks detectable MYH4 expression at both protein and mRNA levels under normal physiological conditions . This fundamental difference represents an evolutionary divergence in muscle fiber type distribution between humans and rodents. Recent research has demonstrated that the capacity for MYH4 expression remains genetically preserved in human muscle cells, as evidenced by successful experimental induction through large MAF transcription factor overexpression . This evolutionary silencing of MYH4 in human muscle underscores the importance of appropriate model selection when studying muscle fiber type-specific processes.

How can researchers optimize detection of experimentally induced MYH4 in human myotubes?

Detecting experimentally induced MYH4 in human myotubes requires a multi-modal approach optimized for maximum sensitivity. For immunofluorescence detection using FITC-conjugated MYH4 antibodies, researchers should implement a carefully optimized protocol: fix differentiated myotubes with 4% paraformaldehyde for 15 minutes, followed by permeabilization with 0.2% Triton X-100 for 10 minutes. Extended blocking (2 hours) with 5-10% normal serum should precede antibody incubation. When working with FITC-conjugated MYH4 antibodies, dilution optimization is essential, typically testing ranges from 1:50 to 1:500 . For verification of successful MYH4 induction, complement immunofluorescence with qPCR analysis quantifying fold-change in MYH4 transcript levels, with primers designed to amplify human-specific MYH4 sequences . Additionally, RNA-bound ribosome isolation techniques such as the AHARIBO RNA system can assess whether MYH4 mRNA is actively translated, providing evidence of protein synthesis potential . For definitive verification of protein translation, LC-MS/MS analysis following immunoprecipitation can identify MYH4-specific peptide signatures that distinguish it from other myosin heavy chain isoforms .

What experimental approaches can verify the specificity of MYH4 antibody reactions in human skeletal muscle?

Verifying MYH4 antibody specificity in human skeletal muscle requires a comprehensive validation strategy. Begin with western blot analysis comparing human and rodent (mouse, rat) skeletal muscle lysates, expecting strong bands at 223-230 kDa in rodent samples but minimal to no detection in normal human samples unless experimentally modified . Implement peptide competition assays by pre-incubating the antibody with purified MYH4 antigenic peptide before application to tissues; specific binding should be blocked by this competition. Cross-reactivity assessment is essential through parallel testing against recombinant MYH1, MYH2, and MYH7 proteins, as these myosin heavy chain isoforms share structural similarities with MYH4. For experimentally induced MYH4 expression systems (e.g., MAF-overexpressing human myotubes), correlation between antibody staining patterns and MYH4 mRNA expression measured by qPCR provides validation of detection specificity . Mass spectrometry validation offers definitive specificity confirmation—samples immunoprecipitated with MYH4 antibodies should yield MYH4-specific peptides identifiable by LC-MS/MS, as demonstrated in recent research with MAFB and MAF-overexpressing human myotubes .

How can FITC-conjugated MYH4 antibodies be integrated into multiplex immunofluorescence protocols?

Integration of FITC-conjugated MYH4 antibodies into multiplex immunofluorescence requires careful consideration of spectral compatibility and staining protocol design. When designing multiplex panels, select additional fluorophores with minimal spectral overlap with FITC (excitation ~495nm, emission ~520nm) such as Cy3 (emission ~570nm), Alexa Fluor 594 (emission ~617nm), or Cy5 (emission ~670nm) . Implement sequential staining protocols for optimal results, applying the FITC-conjugated MYH4 antibody last in the sequence to minimize potential interference. For muscle sections, include robust blocking steps using 5-10% normal serum from species unrelated to any primary antibodies plus 1% BSA to minimize background . Carefully titrate the FITC-conjugated MYH4 antibody concentration, typically testing dilutions from 1:50 to 1:500 to determine optimal signal-to-noise ratio in your specific tissue context . Include appropriate controls with each experiment: no-primary antibody controls to assess secondary antibody specificity, and single-color controls for each fluorophore to verify absence of spectral bleed-through. For long-duration imaging experiments or time-lapse studies, employ anti-fade mounting media specifically formulated to preserve FITC signal and minimize photobleaching during extended microscopy sessions.

How should researchers optimize antigen retrieval for MYH4 detection in formalin-fixed paraffin-embedded tissue?

Optimizing antigen retrieval is critical for successful MYH4 detection in formalin-fixed paraffin-embedded (FFPE) tissue sections. Based on published protocols, heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) has demonstrated superior results for MYH4 epitope exposure . This approach effectively breaks protein cross-links formed during fixation while preserving tissue morphology. The retrieval buffer should be prepared freshly by combining 10mM Tris Base, 1mM EDTA, and 0.05% Tween 20, with pH carefully adjusted to 9.0. For consistent results, researchers should use calibrated retrieval systems (pressure cooker or automated retrieval instruments) rather than microwave methods, with optimal retrieval temperatures of 95-97°C maintained for 20-30 minutes . As an alternative method, citrate buffer (pH 6.0) can be employed, though this typically yields lower staining intensity for MYH4 . Section thickness significantly impacts retrieval efficiency, with 4-5μm sections generally providing optimal results for balancing antibody penetration and tissue integrity. Following retrieval, a 20-minute cooling period at room temperature allows gradual tissue temperature normalization before proceeding with immunostaining. For comparative studies examining multiple myosin heavy chain isoforms, it is essential to standardize the retrieval method across all antibodies to ensure consistent epitope exposure.

What strategies can maximize signal-to-noise ratio when using FITC-conjugated MYH4 antibodies?

Maximizing signal-to-noise ratio with FITC-conjugated MYH4 antibodies requires a comprehensive approach addressing multiple technical aspects. Begin with sample preparation optimization: for frozen sections, use fresh tissue snap-frozen in isopentane cooled with liquid nitrogen, and maintain section thickness at 8-10μm for optimal antibody penetration; for cultured cells, fix with 4% paraformaldehyde for precisely 10-15 minutes to preserve epitope accessibility while maintaining cellular structure. Implement stringent blocking procedures using 5% normal serum from a species unrelated to the host of antibody production, supplemented with 1% BSA and 0.3% Triton X-100, applied for a minimum of 60 minutes . For antibody application, determine optimal concentration through careful titration experiments, typically testing dilutions from 1:50 to 1:500 for FITC-conjugated antibodies . Extend primary antibody incubation times to 12-18 hours at 4°C to enhance specific binding while minimizing background. Address autofluorescence issues, particularly problematic in muscle tissue, by implementing a 10-minute pre-treatment with 0.1% Sudan Black B in 70% ethanol before antibody application. During image acquisition, optimize microscope settings specifically for FITC: use narrow bandpass excitation/emission filters to minimize autofluorescence detection, adjust exposure times to prevent saturation while maximizing signal, and implement post-acquisition processing techniques such as deconvolution or background subtraction to further enhance contrast.

How should researchers interpret MYH4 expression data across different experimental models?

Interpreting MYH4 expression data requires a nuanced understanding of species-specific and experimental context differences. In rodent models, robust MYH4 expression is normal, with protein readily detectable in skeletal muscle, particularly in fast-glycolytic fiber-rich muscles . Conversely, in human skeletal muscle, native MYH4 expression is typically undetectable at protein level and minimal at mRNA level under normal physiological conditions . Therefore, any detectable MYH4 signal in unmanipulated human samples should be carefully validated to rule out antibody cross-reactivity with other myosin heavy chains. Recent research has demonstrated that MYH4 expression can be experimentally induced in human muscle cells through overexpression of large MAF transcription factors, increasing expression 100-1000 fold compared to controls . In these experimental systems, MYH4 mRNA is not only transcribed but also actively translated, as evidenced by ribosome-associated mRNA isolation and LC-MS/MS protein detection . Importantly, correlation analyses from human muscle biopsy samples have revealed that endogenous large MAF expression levels positively correlate with MYH4 and MYH1 (fast fiber genes) expression and negatively correlate with MYH7 (slow fiber gene) expression . These findings suggest that while MYH4 is evolutionarily silenced in human muscle, the regulatory machinery remains functional and responsive to transcription factor manipulation.

How can researchers accurately quantify MYH4-positive fibers in immunofluorescence images?

Accurate quantification of MYH4-positive fibers in immunofluorescence images requires systematic image analysis methodology. Begin with standardized image acquisition: capture multiple representative fields (minimum 5-10 per sample) using consistent exposure parameters, and include calibration standards in each imaging session to normalize fluorescence intensity across experiments. For automated analysis, implement a multi-step image processing workflow: apply background subtraction algorithms to remove uneven illumination artifacts, followed by adaptive thresholding to segment muscle fibers from interstitial space. Use automated fiber detection algorithms that delineate individual fiber boundaries based on membrane markers or negative space between fibers. Define objective criteria for MYH4-positivity using fluorescence intensity thresholds, typically set at 2-3 standard deviations above background or through comparison with positive control samples (rodent muscle sections). For each detected fiber, extract multiple parameters including cross-sectional area, mean fluorescence intensity, and integrated density (product of area and mean intensity). Classify fibers as MYH4-positive or negative based on established threshold criteria, and calculate the percentage of positive fibers relative to total fiber count. For more sophisticated analysis, implement co-localization studies with other myosin heavy chain isoforms to create comprehensive fiber type profiles. Ensure statistical robustness by analyzing sufficient fiber numbers (minimum 200-500 fibers per sample) and implementing appropriate statistical tests for comparing distributions between experimental groups.

What considerations are important when correlating MYH4 expression with functional muscle characteristics?

Correlating MYH4 expression with functional muscle characteristics requires integrated analysis across multiple experimental dimensions. When designing such studies, researchers must account for species-specific expression patterns – MYH4 expression in rodents reflects normal physiology, while in humans, detectable expression typically represents experimental manipulation or potentially pathological states . For meaningful correlation analysis, muscle functional parameters should be collected in parallel with molecular data, including contractile properties (force generation, contraction/relaxation velocities), fiber type distribution assessed through comprehensive myosin heavy chain profiling, metabolic characteristics (glycolytic enzyme activities, mitochondrial content), and fatigue resistance properties. Recent research has established that large MAF transcription factors not only induce MYH4 expression but also profoundly alter the expression of genes associated with glycolytic metabolism, enhancing glycolytic activity in human skeletal muscle cells . This suggests that MYH4 expression serves as part of a coordinated metabolic program rather than an isolated molecular event. When analyzing correlations, researchers should implement multivariate statistical approaches that can account for the complex relationships between molecular expression patterns and functional outcomes. Additionally, longitudinal studies examining how MYH4 expression changes correlate with functional adaptations over time provide more informative insights than single time-point analyses. For translational relevance, researchers should consider how experimentally induced MYH4 expression in human muscle models might relate to therapeutic applications targeting muscle function in conditions characterized by fiber type alterations or metabolic dysfunction.