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MYH4 / Myosin-4 · IHC design guide
Design Immunohistochemistry for MYH4
Plan chromogenic MYH4 IHC in paraffin sections around cytoplasmic staining in skeletal and heart muscle (HPA tissue IHC). Keep fixation consistent (standard IHC practice) and interpret staining with the reported multi-gene targeting caution (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
MYH4 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 in skeletal and heart muscle (HPA tissue IHC) | |
| Staining pattern | Myocyte and cardiomyocyte cytoplasm; medium staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A09143-1) | |
| 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 across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Antibody may target proteins from more than one gene (HPA tissue IHC) | |
| Regulation | No expression regulators annotated (UniProt) | |
| Isoform / epitope | No annotated isoforms or chain cleavage; full-length protein (UniProt) |
Section 1
Recommended MYH4 IHC & IF Protocols
The catalog antibody uses EDTA retrieval (datasheet: A09143-1). Published IHC protocols cover dog frozen muscle sections (PMC11672843) and human eyelid FFPE sections (PMC11802858).
| Sample | Paraffin-embedded mouse skeletal muscle tissue; fixative not specified (datasheet A09143-1) |
| Fixation | Image fixative and duration unreported (datasheet A09143-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A09143-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A09143-1) |
| Primary antibody | Rabbit anti-MYH4, 1:50 recommended; image 1:100 (datasheet A09143-1) |
| Primary incubation | Overnight at 4 °C (datasheet A09143-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A09143-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | MYH4-positive staining in cardiomyocytes of heart muscle (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in skeletal muscle and heart muscle. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet: A09143-1). The published eyelid FFPE protocol used pH 6.0 (PMC11802858).
Section 2
What Is the Expected MYH4 Staining Pattern?
MYH4 is a cytoplasmic myofibrillar protein of muscle thick filaments and has no transmembrane segment (UniProt Q9Y623). In paraffin section IHC, expect staining in skeletal myocytes and cardiomyocytes (HPA: Medium in both). Interpret that pattern cautiously: HPA rates tissue IHC as Supported, reports medium consistency with RNA data, and warns that the antibody staining may represent proteins from more than one gene.
| Cytoplasmic staining within skeletal myocytes or cardiomyocytes, with surrounding structures comparatively clear. | This fits the reported Medium staining in both cell types (HPA tissue IHC) and MYH4's location in myofibrillar thick filaments (UniProt Q9Y623). The observed pattern supports a muscle associated signal; it does not by itself resolve the antibody's multi gene specificity warning (HPA tissue IHC). |
| Predominantly nuclear or sharply cell surface staining, without a convincing cytoplasmic muscle signal. | Treat this as discordant with the reported cytoplasmic pattern (HPA tissue IHC) and myofibrillar location without a transmembrane segment (UniProt Q9Y623). Review morphology and controls before scoring it as MYH4; the compartment alone cannot identify which staining step caused the discrepancy. |
| Prominent staining in cells reported as negative, such as adipocytes or adrenal glandular cells. | HPA reports these cells as Not detected (HPA tissue IHC). Check for antibody cross reaction and detection related signal using suitable controls (general IHC practice). HPA's warning that staining may represent proteins from more than one gene limits any claim that unexpected staining is MYH4 specific. |
| Widespread, low contrast color across muscle and nonmuscle areas, with little cellular definition. | Diffuse background is difficult to reconcile with the cell associated cytoplasmic pattern (HPA tissue IHC). Uneven blocking, excessive detection signal, or inadequate washing can produce background in chromogenic IHC (general IHC practice); appearance alone cannot distinguish these causes. |
| No staining in a skeletal muscle or heart muscle section that serves as the positive tissue. | Both tissues have reported Medium staining, so an absent signal calls for a run check (HPA tissue IHC). Inspect tissue preservation and assay controls, then review retrieval, antibody conditions, and detection in that order as appropriate (general IHC practice). Do not infer a MYH4 specific fixation effect. |
💡Expected MYH4 appearanceCall a section positive when myocyte or cardiomyocyte cytoplasm shows convincing Medium muscle associated staining (HPA tissue IHC; UniProt Q9Y623); isolated nuclear, cell surface, or negative cell staining is discordant with that reference pattern (HPA tissue IHC; UniProt Q9Y623).
| Compartment and muscle structure | MYH4 resides in cytoplasmic myofibrillar thick filaments and lacks a transmembrane segment (UniProt Q9Y623). Use muscle cell cytoplasm as the anatomical reference when scoring IHC; a membrane or nuclear dominant pattern needs independent scrutiny. |
| Reference tissue distribution | HPA reports Medium staining in skeletal myocytes and cardiomyocytes and Not detected staining in the listed negative cell types (HPA tissue IHC). Its RNA summary is tissue enriched in skeletal muscle (HPA tissue IHC); that summary does not negate the reported heart IHC signal. |
| Antibody level confidence | The listed antibody HPA001349 is IHC Supported, while HPA describes medium agreement with RNA and cautions that staining may represent more than one gene (HPA antibody record; HPA tissue IHC). Score a compatible pattern as supportive evidence, with specificity interpreted cautiously. |
| IF or ICC interpretation | For the separate IF or ICC guide: is focal adhesion staining the expected MYH4 readout? HPA lists focal adhesion sites as uncertain and the antibody's ICC status as Uncertain (HPA subcellular; HPA antibody record). That observation cannot replace the tissue IHC cytoplasmic reference. |
| Situation | Likely cause | Next action |
|---|---|---|
| The positive muscle control is completely blank. | An assay or sample problem is plausible because HPA reports Medium staining in skeletal myocytes and cardiomyocytes (HPA tissue IHC); the blank section does not identify a target specific cause. | Confirm tissue identity and morphology, then check reagent delivery, retrieval record, antibody application, and detection controls (general IHC practice). Record the failed control before interpreting test sections. |
| Muscle staining is faint or uneven despite an intact positive control. | Section variation or uneven assay exposure can change visible chromogenic signal (general IHC practice). HPA's Medium reference is a qualitative observation, not a required intensity threshold (HPA tissue IHC). | Compare matched section areas and assay controls; review section quality, coverage, and documented retrieval and detection settings (general IHC practice). Avoid labeling a faint area negative solely by comparison with the darkest field. |
| Nuclei or cell outlines dominate the staining. | That distribution conflicts with cytoplasmic muscle staining (HPA tissue IHC) and MYH4's myofibrillar, nontransmembrane location (UniProt Q9Y623). A particular procedural cause cannot be assigned from the pattern alone. | Recheck cell boundaries against the counterstain and inspect negative controls (general IHC practice). Score the dominant discordant pattern separately from any convincing myocyte cytoplasmic signal. |
| Color appears in reported negative cells or without primary antibody. | HPA lists adipocytes and adrenal glandular cells as Not detected (HPA tissue IHC). Signal without primary antibody suggests detection background; staining confined to the full assay may also involve antibody cross reaction (general IHC practice). | Compare the no primary control, inspect endogenous chromogen or enzyme activity, and review blocking and detection conditions (general IHC practice). Keep HPA's multi gene antibody warning in the specificity assessment. |
| A uniform haze obscures otherwise plausible muscle staining. | Low contrast background can arise from nonspecific reagent binding or excessive detection development (general IHC practice); HPA's reference pattern is cellular and cytoplasmic (HPA tissue IHC). | Compare negative controls, review antibody concentration and detection time, and assess wash and blocking steps (general IHC practice). Reassess cell localization only after the background is controlled. |
| ICC or IF shows focal adhesion staining instead of the tissue IHC pattern. | HPA calls the focal adhesion assignment uncertain, notes antibodies may target more than one gene, and rates the listed antibody ICC Uncertain (HPA subcellular; HPA antibody record). | Interpret the IF or ICC result under its separate guide and validation controls; do not use it to redefine the cytoplasmic muscle IHC reference (HPA tissue IHC; HPA subcellular). |
Experiment evidence
IHC & IF images showing MYH4 staining
A09143-1 · IHCIHC analysis of MYH4 using anti-MYH4 antibody (A09143-1). MYH4 was detected in a paraffin-embedded section of mouse skeletal muscle tissue.
A09143-1 · IHCIHC analysis of MYH4 using anti-MYH4 antibody (A09143-1). MYH4 was detected in a paraffin-embedded section of rat skeletal muscle tissue. He
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000264424-MYH4/tissue/cerebral+cortex#imid_489374
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000264424-MYH4/tissue/colon#imid_489308
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000264424-MYH4/tissue/liver#imid_489301
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000264424-MYH4/tissue/kidney#imid_489294
HPA · ICC-IF OE19Human Protein Atlas immunofluorescence, OE19, antibody HPA001349; green = MYH4, blue = nucleus, red = microtubules.
HPA · ICC-IF OE19Human Protein Atlas immunofluorescence, OE19, antibody HPA001349; green = MYH4, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for MYH4 IHC & IF
🧪Run skeletal muscle first: myocytes should show MYH4 staining (HPA: Medium in skeletal muscle myocytes). Use adipose tissue as the negative tissue (HPA: Not detected in adipocytes); interstitial nonmuscle cells on the skeletal muscle slide should lack myofibrillar staining (UniProt Q9Y623: myofibril localization).
Positive control tissue: Heart muscle (Cardiomyocytes, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show MYH4 in OE19, SuSa, U2OS, with annotated localisation: Focal adhesion sites (uncertain) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a rabbit IgG isotype control matched to the primary antibody’s clonality, and MYH4-knockout tissue as a biological negative (caption: rabbit anti-MYH4 primary). For chromogenic IHC, quench endogenous peroxidase and check for background in skeletal muscle; for IF, assess muscle autofluorescence (caption: peroxidase secondary and DAB detection).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A09143-1 tissue-IHC caption does not state a fixative. The paraffin-section example used heat-mediated EDTA retrieval at pH 8.0 and primary antibody at 1:100, but it does not establish whether frozen sections or IF are easier (A09143-1 tissue-IHC caption). Skeletal muscle autofluorescence can complicate IF interpretation; assess it with a no-primary control.
HPA tissue IHC evidence for MYH4
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — Medium consistency between antibody staining and RNA expression data. Caution, targets protein from more than one gene.). 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 MYH4 IHC Tips
Troubleshoot MYH4 staining in paraffin sections by checking retrieval, intracellular localisation, controls and scoring against the reported skeletal muscle IHC result (datasheet A09143-1).
Which retrieval conditions should I try first when MYH4 staining is weak?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A09143-1). The documented mouse skeletal muscle result used this retrieval, followed by 10% goat serum blocking and antibody at 1:100 overnight at 4°C (datasheet A09143-1). If staining remains weak, vary heating duration on matched sections while holding antibody concentration and detection conditions constant, then compare myocyte staining with background (standard IHC practice; HPA: skeletal muscle myocytes Medium). Record the retrieval duration and tissue condition for each slide, because excessive heating can damage morphology and make cytoplasmic staining difficult to interpret (standard IHC practice; UniProt Q9Y623: myofibril localisation).
How should I investigate weak staining when the tissue fixative is uncertain?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative (datasheet A09143-1). Record the actual fixative and fixation duration for each specimen, then compare sections processed with the same EDTA pH 8.0 retrieval and 1:100 primary antibody conditions used in the documented result (standard IHC practice; datasheet A09143-1). Include a skeletal muscle control in each run and assess both myocyte signal and preserved morphology before changing detection conditions (HPA: skeletal muscle myocytes Medium; standard IHC practice). Do not attribute a weak result specifically to fixation without a controlled comparison of specimens, since the caption supplies no such comparison (datasheet A09143-1).
Where should convincing MYH4 staining appear in a muscle section?
Expect intracellular staining associated with myofibrils and their thick filaments, rather than a cell-surface outline (UniProt Q9Y623: cytoplasm and myofibril localisation; UniProt Q9Y623: no transmembrane segment). In tissue IHC, cytoplasmic staining is reported in skeletal muscle myocytes and heart muscle cardiomyocytes, each at Medium level (HPA: tissue IHC profile). Compare the staining with fibre morphology and a matched negative control before calling diffuse deposit or extracellular colour positive (standard IHC practice; UniProt Q9Y623: myofibril localisation). The reported focal-adhesion localisation is uncertain and comes with a warning that its antibodies target proteins from multiple genes, so it should not override the muscle-section pattern (HPA: subcellular localisation).
Can this IHC signal be assigned specifically to MYH4 rather than related myosins?
The record lists 0 alternative isoforms for MYH4, but that alone does not establish antibody specificity among myosin heavy chains (UniProt Q9Y623: isoforms and protein identity). HPA rates its tissue staining Supported while warning that its antibodies target proteins from more than one gene, so interpret its muscle pattern cautiously (HPA: tissue IHC reliability). MYH4 contains a motor domain at residues 86–782 and an IQ region at 785–814; the supplied evidence does not map the catalog antibody’s epitope to either region (UniProt Q9Y623: domains; datasheet A09143-1: caption). For a gene-specific conclusion, seek independently established antibody specificity and compare expected myofibrillar staining with appropriate tissue controls (standard IHC practice; UniProt Q9Y623: myofibril localisation).
How should I assess MYH4 in a multiplex IF experiment?
Pair MYH4 with an independently validated myocyte marker and inspect each fluorescence channel separately before interpreting overlap (HPA: skeletal muscle myocytes Medium; standard IF practice). Choose a fluorophore and filter combination that separates MYH4 signal from tissue autofluorescence, and include unstained and secondary-only controls for the same tissue (standard IF practice). Because MYH4 is intracellular in myofibrils and has no transmembrane segment, optimise permeabilisation for access to the intracellular epitope while preserving fibre structure (UniProt Q9Y623: localisation and topology; standard IF practice). The documented 1:100 concentration and EDTA pH 8.0 retrieval belong to paraffin-section chromogenic IHC; establish IF conditions independently rather than treating that caption as IF validation (datasheet A09143-1).
What should I check when DAB obscures MYH4 staining?
The documented paraffin-section IHC used 10% goat serum blocking, a 1:100 rabbit primary overnight at 4°C, and peroxidase-linked detection with DAB (datasheet A09143-1). If colour appears throughout the section, compare no-primary and secondary-only controls, confirm adequate washing, and check whether DAB development is excessive (standard IHC practice). Include a peroxidase-blocking step when using HRP detection, and assess endogenous enzyme colour against the controls before assigning it to MYH4 (standard IHC practice). Evaluate specific signal in myocyte cytoplasm against nearby unstained structures and negative-control tissue, since MYH4 is associated with myofibrils and HPA reports many nonmuscle tissues as Not detected (UniProt Q9Y623: myofibril localisation; HPA: tissue IHC).
How can I quantify MYH4 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define myocytes as the scoring compartment and exclude damaged or folded regions before measuring chromogenic signal (HPA: skeletal muscle myocytes Medium; standard IHC practice). Report the percentage of positive myocytes and staining intensity, or use an H-score from 0–300 based on the percentages at intensity grades 0–3 (standard IHC practice). If measuring stained area instead, normalise DAB-positive area to evaluable myocyte area in mm², using the same threshold and counterstain handling across slides (standard IHC practice). Keep retrieval, antibody incubation and DAB development consistent, then report biological specimens separately from fields sampled within each specimen (datasheet A09143-1: documented workflow; standard IHC practice).
How do I distinguish true MYH4 signal from staining artefacts?
A convincing result follows myocyte cytoplasm and myofibrils in preserved muscle fibres, with an appropriate negative-control result (UniProt Q9Y623: myofibril localisation; HPA: skeletal muscle myocytes Medium; standard IHC practice). Question staining confined to cell borders or extracellular space, because the recorded protein has no transmembrane segment and resides in myofibrillar thick filaments (UniProt Q9Y623: topology and localisation). Also flag section-edge intensification, necrotic regions and widespread DAB colour as possible processing or endogenous-enzyme artefacts, then check matched controls (standard IHC practice). Heart muscle staining can be compatible with HPA’s reported cytoplasmic pattern, but its Supported rating includes a warning about antibodies targeting proteins from multiple genes (HPA: tissue IHC reliability and profile).
Boster reagents
Best MYH4 / Myosin-4 IHC Antibodies
A09143-1 has IHC images from paraffin-embedded mouse and rat skeletal muscle sections (A09143-1 IHC captions). Human reactivity is listed, but no IF image is supplied (A09143-1 catalog reactivity; IF image alts).
Real IHC data
Anti-MYH4 Antibody
Cat # A09143-1
A09143-1 is listed for IHC and has a mouse skeletal muscle paraffin-section image (A09143-1 catalog applications; mouse IHC caption). Its second image shows rat skeletal muscle paraffin sections; human reactivity is listed without a human IHC image (A09143-1 rat IHC caption; catalog reactivity; IHC image alts).
Which to pick: For tissue IHC, choose A09143-1 based on its mouse and rat paraffin-section images; the fixative is unreported (A09143-1 IHC captions). For IF/ICC, this catalog provides no validated option: A09143-1 has no listed IF/ICC application or IF image (A09143-1 catalog applications; IF image alts). For cross-species IHC, A09143-1 is a rabbit polyclonal listed as reactive with human, mouse, and rat, with direct IHC images for mouse and rat only (A09143-1 catalog host/dilution_raw/reactivity; IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9Y623 (MYH4_HUMAN, Myosin-4).
- Human Protein Atlas. MYH4 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. MYH4 subcellular location (ICC-IF): Localized to the focal adhesion sites. Caution: Based on antibodies targeting proteins from multiple genes..
- Human Protein Atlas. MYH4 antibody validation summary (1 antibodies).
- High expression of HILPDA is an adverse prognostic prognostic factor in hepatocellular carcinoma. Medicine 2023 — PMC9981432.
- Comparative Analysis of Muscle Fibers in Selected Muscles of Working and Companion Dog Breeds. Animals : an open access journal from MDPI 2024 — PMC11672843.
- Creation of knockin mice for the fluorescence protein based in vivo identification of skeletal myofiber types. Scientific reports 2025 — PMC11968920.
- Age-related thinning of orbicularis oculi muscle inside upper eyelid and its possible association with sunken upper eyelids. Scientific reports 2025 — PMC11802858.
- PubMed PMID:10388558 — UniProt-cited evidence.
- PubMed PMID:16625196 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for MYH4 (Myosin-4). Expected localisation Cytoplasm in skeletal and heart muscle (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A09143-1). Validated IHC antibody: A09143-1 at 1:50 recommended; image 1:100 (the product's own IHC validation image is shown). Controls (HPA): positive Heart muscle (Cardiomyocytes); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded mouse skeletal muscle tissue; fixative not specified (datasheet A09143-1); 2. Fixation: Image fixative and duration unreported (datasheet A09143-1); 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 retrieval: EDTA pH 8.0 (datasheet A09143-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A09143-1); 8. Primary antibody: Rabbit anti-MYH4, 1:50 recommended; image 1:100 (datasheet A09143-1); 9. Primary incubation: Overnight at 4 °C (datasheet A09143-1). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Antibody may target proteins from more than one gene (HPA tissue IHC); Isoform / epitope: No annotated isoforms or chain cleavage; full-length protein (UniProt); Regulation: No expression regulators annotated (UniProt). Evidence: UniProt Q9Y623, HPA tissue IHC · Supported, HPA ICC-IF · focal adhesion sites. Caution: Based on antibodies targeting…, Datasheet A09143-1, PMC11672843, PMC11802858. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/MYH4/immunohistochemistry-ihc-guide.