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MYH3 / Myosin-3 · IHC design guide
Design Immunohistochemistry for MYH3
Plan chromogenic MYH3 IHC-P around medium staining in cardiomyocytes and skeletal myocytes (HPA tissue IHC). Start the catalog antibody at 5 μg/mL (datasheet: A02697), and treat cytoplasmic myofibrillar localization as a molecular expectation (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
MYH3 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasm; myofibrillar thick filaments (UniProt) | |
| Staining pattern | Medium staining in cardiomyocytes and skeletal myocytes (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Heart muscle+1 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent between sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Fetal tissue expression documented (UniProt) | |
| Isoform / epitope | 0 isoforms; one cytoplasmic chain spanning residues 1–1940 (UniProt) |
Section 1
Recommended MYH3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by one published MYH3 chromogenic IHC protocol in rabbit skeletal muscle (PMC10340610).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A02697); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-MYH3, 5 μg/mL (datasheet A02697) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | MYH3-positive staining in cardiomyocytes of heart muscle (HPA tissue IHC: Medium). HPA tissue profile: Expression in skeletal and heart muscle. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page retrieval); consider the published autoclave retrieval for rabbit muscle (PMC10340610).
Section 2
What Is the Expected MYH3 Staining Pattern?
MYH3 should appear in the cytoplasm, along the myofibrils’ thick filaments (UniProt P11055: subcellular location). In tissue IHC, expect staining in skeletal myocytes and heart cardiomyocytes; HPA reports medium staining in both and rates its tissue pattern Enhanced, while noting medium consistency with RNA data (HPA: tissue IHC). MYH3 has no transmembrane segment, so a membrane-only pattern would be unexpected (UniProt P11055: topology).
| Myocytes or cardiomyocytes show cytoplasmic staining that follows the contractile tissue architecture. | This fits MYH3’s myofibrillar location and HPA’s medium staining in skeletal and heart muscle (UniProt P11055: subcellular location; HPA: tissue IHC). Judge the pattern within intact cells rather than treating every brown deposit near a muscle fiber as intracellular signal (general IHC practice). |
| A crisp membrane rim or predominantly nuclear staining appears in otherwise intact muscle cells. | Neither compartment matches the reported cytoplasmic thick-filament location; MYH3 also lacks a transmembrane segment (UniProt P11055: subcellular location and topology). Review tissue morphology and the detection controls before interpreting this as MYH3 expression (general IHC practice). |
| Strong staining appears mainly in adipocytes or glandular cells outside muscle. | These are suspect assignments: HPA reports MYH3 as not detected in adipocytes from adipose tissue and glandular cells from adrenal gland or appendix (HPA: tissue IHC). Check for cross-reactivity and, when using enzyme detection, endogenous activity (general IHC practice). |
| Color is spread across the section, including spaces between cells, without a clear myocyte pattern. | Diffuse deposits do not establish the myofibrillar location reported for MYH3 (UniProt P11055: subcellular location). Compare a reagent control and inspect washing, blocking, and detection conditions; those are general background checks, not documented MYH3-specific effects (general IHC practice). |
| A skeletal or heart muscle section has no detectable myocyte or cardiomyocyte staining. | HPA reports medium staining in those cells, so an absent signal warrants a technical review (HPA: tissue IHC). It does not alone prove that the specimen lacks MYH3: compare a verified positive section and assess morphology, antibody incubation, and detection performance (general IHC practice). |
💡Expected MYH3 appearanceCall a result positive when skeletal myocytes or heart cardiomyocytes show predominantly cytoplasmic, myofibril-associated staining around HPA’s reported medium level; isolated membrane, nuclear, or nonmuscle deposits are suspect (UniProt P11055: subcellular location and topology; HPA: tissue IHC).
| Cell type and tissue | HPA observes medium MYH3 staining in skeletal myocytes and heart cardiomyocytes, with several listed nonmuscle cell types not detected (HPA: tissue IHC). Use the cell carrying the stain, not the tissue label alone, when judging agreement. |
| Antibody evidence | HPA rates the tissue IHC pattern Enhanced and records IHC Enhanced for antibody HPA021808, while describing only medium consistency between staining and RNA data (HPA: tissue IHC; HPA: antibodies). This supports the reported pattern without making every unexpected deposit specific. |
| Intracellular location and topology | MYH3 is reported in cytoplasmic myofibril thick filaments and has no transmembrane segment (UniProt P11055: subcellular location and topology). Interpret a membrane-only pattern cautiously; the supplied record does not identify an antibody epitope or a target-specific retrieval condition. |
| Developmental expression context | UniProt reports expression in fetal bone, thymus, placenta, heart, brain, and liver (UniProt P11055: tissue specificity). That statement does not predict staining intensity in a particular section; use HPA’s observed skeletal and heart muscle IHC pattern for the comparison here (HPA: tissue IHC). |
| IF/ICC evidence? | HPA supplies no ICC-IF image or subcellular location assignment for MYH3 in its ICC-IF record (HPA: subcellular). The cytoplasmic myofibrillar expectation comes from UniProt, while the medium tissue intensity comes from IHC; neither establishes an IF staining intensity (UniProt P11055: subcellular location; HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| No chromogenic signal appears in a skeletal or heart muscle positive section. | The observed result conflicts with HPA’s medium staining in myocytes or cardiomyocytes; the section alone cannot distinguish low antigen from a failed staining run (HPA: tissue IHC; general IHC practice). | Run a verified positive section alongside it, confirm tissue preservation and antibody and detection steps, then review retrieval against the antibody’s validated IHC instructions (general IHC practice). No MYH3-specific retrieval condition is supplied. |
| Signal is weak or patchy within the expected muscle cells. | HPA reports medium staining, not uniformly strong staining (HPA: tissue IHC). Section quality, incubation, or detection can also affect visible signal in IHC generally (general IHC practice). | Compare intact regions and a concurrently stained positive section before scoring; review the antibody’s validated IHC instructions and detection controls (general IHC practice). Do not infer a MYH3-specific fixation effect from this pattern. |
| A nuclear or membrane-only pattern dominates. | That distribution conflicts with cytoplasmic myofibril thick filaments and the absence of a transmembrane segment (UniProt P11055: subcellular location and topology). | Inspect cell boundaries and tissue morphology, then compare the primary-omission control and a known-positive muscle section to locate nonspecific deposition (general IHC practice). |
| Adipocytes or glandular cells stain more strongly than muscle cells. | HPA lists several sampled adipocyte and glandular cell populations as not detected; cross-reactivity or endogenous detection activity becomes a concern (HPA: tissue IHC; general IHC practice). | Check a primary-omission control and the detection system; if using peroxidase chemistry, assess endogenous peroxidase blocking before assigning the signal to MYH3 (general IHC practice). |
| Diffuse color covers cells and extracellular spaces. | A widespread deposit lacks the cell and compartment pattern expected from HPA tissue staining and UniProt myofibrillar localization (HPA: tissue IHC; UniProt P11055: subcellular location). | Review blocking, wash steps, antibody concentration, and chromogen development with reagent controls; change one general IHC variable at a time and compare the muscle-cell pattern (general IHC practice). |
| IF/ICC signal is present, but its location is uncertain. | HPA provides no MYH3 ICC-IF images or assigned ICC-IF location in this payload, so its tissue IHC intensity cannot validate an IF pattern (HPA: subcellular; HPA: tissue IHC). | Assess IF with its own controls and compare intracellular localization against UniProt’s cytoplasmic myofibrillar assignment; use the dedicated IF/ICC guide for its protocol (UniProt P11055: subcellular location; general IF practice). |
Experiment evidence
IHC & IF images showing MYH3 staining
A02697 · IHCImmunohistochemistry of MYH3 in mouse skeletal muscle tissue with MYH3 antibody at 5 μg/ml.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000109063-MYH3/tissue/cerebral+cortex#imid_5822620
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000109063-MYH3/tissue/colon#imid_5815778
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000109063-MYH3/tissue/liver#imid_5815771
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000109063-MYH3/tissue/kidney#imid_5815764
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for MYH3 IHC & IF
🧪Run skeletal muscle first: myocytes should show MYH3 staining (HPA: Medium in skeletal-muscle myocytes). Use adipose tissue as the negative tissue (HPA: Not detected in adipocytes); on the skeletal-muscle slide, nuclei and interstitial areas should lack specific myofibrillar staining (UniProt P11055: myofibrillar thick-filament localisation).
Positive control tissue: Heart muscle (Cardiomyocytes, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for MYH3; derive a cell-line control from the positive tissue's cell type (Cardiomyocytes) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary (secondary-only) control, an isotype control matched to the primary antibody’s host species and immunoglobulin class, and MYH3-knockout tissue if available (standard IHC practice). For chromogenic staining, block endogenous peroxidase; for IF, assess muscle autofluorescence with an unstained section (standard IHC/IF practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence, and the selected A02697 tissue-IHC caption does not state a fixative (A02697 caption: fixative unreported). Antigen-retrieval dependence is unreported, so optimise retrieval on the intended paraffin sections (standard IHC practice). The evidence does not establish whether frozen sections or IF would be easier; muscle autofluorescence may complicate IF interpretation (standard IF practice).
HPA tissue IHC evidence for MYH3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data. External characterization data supports antibody staining.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Heart muscle | Cardiomyocytes | Medium | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced MYH3 IHC Tips
Troubleshoot MYH3 staining in paraffin sections by checking retrieval, cellular location and muscle cell controls before interpreting chromogenic signal.
How should I retrieve MYH3 in paraffin sections with weak staining?
Start with citrate pH 6.0 HIER at 95–98 °C for 20 min (page retrieval rule: cytoplasmic antigen). After retrieval, compare an identically processed skeletal muscle control with the test section before increasing antibody concentration (HPA: medium staining in skeletal muscle myocytes; standard IHC practice). MYH3 is associated with cytoplasmic myofibrillar thick filaments, so assess staining within muscle fibers rather than at cell membranes (UniProt P11055: subcellular location). If signal remains weak, vary retrieval duration around the starting condition on adjacent sections while holding detection constant (standard IHC optimization). The selected image caption reports 5 µg/ml but does not report fixation (A02697 tissue-IHC caption).
Could fixation explain inconsistent MYH3 staining between paraffin blocks?
MYH3 specific sensitivity to fixation is unknown from the supplied evidence; the selected tissue image does not state its fixative (A02697 tissue-IHC caption). Record each block’s fixative and fixation time, then compare sections processed through the same citrate pH 6.0 retrieval for 20 min (page retrieval rule; standard IHC practice). Include a skeletal muscle control in each staining run to distinguish a block problem from a run problem (HPA: skeletal muscle myocytes show medium staining; standard IHC practice). Poor morphology, uneven staining or staining concentrated at section edges should prompt a processing review (standard IHC practice). Do not infer MYH3 fixation sensitivity from its localization or modification record (UniProt P11055: myofibrillar location and trimethyllysine).
Where should convincing MYH3 chromogenic signal appear?
Expect signal in the cytoplasmic myofibrillar compartment of muscle cells, where MYH3 is assigned to thick filaments (UniProt P11055: subcellular location). Heart cardiomyocytes and skeletal muscle myocytes have reported medium tissue staining, making them useful context for evaluating distribution (HPA: tissue IHC). MYH3 has no annotated transmembrane segment, so a crisp surface rim alone does not fit its recorded topology (UniProt P11055: topology). Inspect stained sections against hematoxylin counterstain to confirm that signal follows intact muscle cells rather than extracellular spaces (standard IHC practice). Compare suspicious nuclear or stromal deposits with a no primary control before calling them MYH3 positive (standard IHC practice).
How can I assess whether the antibody recognizes the intended MYH3 epitope?
First obtain the antibody’s stated immunogen or epitope before assigning any staining pattern to a specific MYH3 region (standard antibody validation practice). The record annotates 0 isoforms and describes a myosin motor region at residues 86–779, but it does not identify this antibody’s binding site (UniProt P11055: isoforms and domains; supplied product evidence). A reported trimethyllysine occurs at residue 130; its effect on this antibody’s staining is unknown without epitope data (UniProt P11055: modified residues). Compare staining in expected muscle cells with a no primary control and an independent antibody, if available (HPA: muscle tissue IHC; standard IHC validation practice). Treat discordant results as a reason to review specificity, retrieval and tissue processing (standard IHC practice).
How should I investigate MYH3 by IF alongside this IHC guide?
Use a muscle cell marker in a separate channel to test whether MYH3 fluorescence falls within the expected cells (HPA: staining in skeletal myocytes and heart cardiomyocytes; standard IF practice). Select fluorophores after imaging an unstained section, since tissue autofluorescence can overlap a chosen detection channel (standard IF practice). MYH3 is cytoplasmic and lacks a transmembrane segment, so permeabilize cells for access to its intracellular epitope while optimizing detergent exposure for morphology (UniProt P11055: localization and topology; standard IF practice). Include single stain and no primary controls to assess channel bleed through and background (standard IF practice). The supplied HPA subcellular record contains no ICC/IF images, so verify this antibody’s IF performance independently (HPA: subcellular record).
What should I check when MYH3 IHC shows diffuse brown background?
Compare the section with a no primary control to identify signal arising from detection reagents or endogenous activity (standard chromogenic IHC practice). For peroxidase based detection, include a peroxidase block before applying the detection system and evaluate DAB development time on a control section (standard chromogenic IHC practice). Inspect whether brown signal follows intact muscle cell cytoplasm; broad extracellular deposits do not match the recorded myofibrillar location (UniProt P11055: subcellular location). If background increases with primary antibody concentration, titrate around the caption’s 5 µg/ml condition using matched sections (A02697 tissue-IHC caption; standard IHC optimization). Keep blocking, washes and counterstaining consistent across comparisons (standard IHC practice).
How should I quantify MYH3 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the muscle cell compartment before scoring, since MYH3 is assigned to myofibrillar thick filaments (UniProt P11055: subcellular location). Report the percentage of positive muscle cells and, when intensity is reproducible, an H score from intensity categories of 0–3 multiplied by their respective percentages (standard IHC scoring practice). Normalize counts to the number of evaluable muscle cells or stained area to the evaluable muscle area, rather than total section area (standard IHC quantification practice). Hold retrieval, illumination, DAB development and image analysis thresholds constant across samples (page retrieval rule; standard IHC practice). Exclude folds, damaged edges and necrotic regions using prespecified morphology criteria (standard IHC quantification practice).
When is apparent MYH3 positivity likely to be an artefact?
Convincing staining should map to intact muscle cell cytoplasm and myofibrils, the recorded location of MYH3 (UniProt P11055: subcellular location). Skeletal myocytes and heart cardiomyocytes provide reported positive tissue contexts; HPA describes medium staining in both (HPA: tissue IHC). Isolated nuclear staining, signal in an unexpected cell type, or staining confined to tissue edges warrants closer control review (UniProt P11055: subcellular location; standard IHC interpretation). Exclude necrotic areas and compare suspected deposits with no primary and peroxidase block controls before attributing brown color to MYH3 (standard chromogenic IHC practice). The HPA tissue result has Enhanced reliability with medium staining and RNA consistency, so interpret borderline patterns cautiously (HPA: reliability description).
Boster reagents
Best MYH3 / Myosin-3 IHC Antibodies
A02697 is listed for IHC-P with human, mouse and rat reactivity (catalog: applications/reactivity); its IHC image shows mouse skeletal muscle (catalog image caption).
Real IHC data
Anti-Myosin-3 MYH3 Antibody
Cat # A02697
A02697 will render with a mouse skeletal muscle IHC image at 5 μg/mL (catalog image caption). It is listed for IHC-P and human, mouse and rat reactivity, with IHC validation reported in mouse samples (catalog: applications/reactivity; datasheet: validation).
Which to pick: Choose A02697 for paraffin-section tissue IHC: IHC-P is listed, and its own image shows mouse skeletal muscle staining at 5 μg/mL (catalog: applications; catalog image caption). The fixative is unreported (catalog image caption). No SKU in this catalog payload lists IF/ICC validation; A02697 lists human, mouse and rat reactivity, but its reported IHC validation is in mouse samples (catalog: applications/reactivity; datasheet: validation).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P11055 (MYH3_HUMAN, Myosin-3).
- Human Protein Atlas. MYH3 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. MYH3 subcellular location (ICC-IF): Highest expression in NB4: 2.6 nTPM.
- Human Protein Atlas. MYH3 antibody validation summary (1 antibodies).
- Non-Syndromic or Anomaly-Associated Genes (MYH3, GREM1, IRF6) and Their Proteins in Unilateral Right Cleft Tissue. International journal of molecular sciences 2026 — PMC13207050.
- Efficacy of Rectal Systemic Administration of Mesenchymal Stem Cells to Injury Sites via the CXCL12/CXCR4 Axis to Promote Regeneration in a Rabbit Skeletal Muscle Injury Model. Cells 2023 — PMC10340610.
- Novel Therapeutic Effects of Non-thermal atmospheric pressure plasma for Muscle Regeneration and Differentiation. Scientific reports 2016 — PMC4923893.
- A human embryonic limb cell atlas resolved in space and time. Nature 2024 — PMC7616500.
- PubMed PMID:2726495 — UniProt-cited evidence.
- PubMed PMID:16625196 — UniProt-cited evidence.
- PubMed PMID:2806546 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for MYH3 (Myosin-3). Expected localisation Cytoplasm; myofibrillar thick filaments (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A02697 at 5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Heart muscle (Cardiomyocytes); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet A02697); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-MYH3, 5 μg/mL (datasheet A02697); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent between sections. (standard IHC practice; not target-specific); Caveat: Staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 0 isoforms; one cytoplasmic chain spanning residues 1–1940 (UniProt); Regulation: Fetal tissue expression documented (UniProt). Evidence: UniProt P11055, HPA tissue IHC · Enhanced, HPA cell-line RNA · Highest expression in NB4: 2.6 nTPM, Datasheet A02697, PMC10340610. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/MYH3/immunohistochemistry-ihc-guide.