MYH1 / Myosin-1 · IHC design guide

Design Immunohistochemistry for MYH1

Plan chromogenic IHC on paraffin sections using skeletal muscle or heart muscle as positive controls (HPA tissue IHC). This guide covers cytoplasmic staining expectations (HPA tissue IHC) and the catalog antibody’s IHC dilution of 1:50–1:200 (datasheet A09420).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for MYH1 (IHC for MYH1): expected localisation Cytoplasm in muscle; myofibrillar thick filaments (HPA tissue IHC; UniProt), antibody A09420, validated IHC image, and IHC protocol steps — Printable **MYH1** IHC protocol sheet — expected localisation Cytoplasm in muscle; myofibrillar thick filaments (HPA tissue IHC; UniProt), antibody A09420, controls and protocol steps. Open the full MYH1 IHC guide →

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MYH1 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 muscle; myofibrillar thick filaments (HPA tissue IHC; UniProt)
	Staining pattern	High cytoplasmic staining in myocytes and cardiomyocytes (HPA tissue IHC)
	Antigen retrieval	Citrate pH 6.0 HIER, heat-mediated (datasheet A09420)
	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 paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific)
	Caveat	Staining may include proteins from more than one gene (HPA tissue IHC)
	Regulation	No expression regulation annotated (UniProt)
	Isoform / epitope	No annotated isoforms; one 1–1939 chain (UniProt)

Section 1

Recommended MYH1 IHC & IF Protocols

The catalog antibody’s IHC-P protocol is followed by four published MYH1 IHC protocols covering the samples and methods reported in their excerpts (datasheet A09420; PMC12331014; PMC13242725; PMC7807096; PMC9105468).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded Human skeletal muscle tissue; fixative not specified (datasheet A09420)
Fixation	Image fixative and duration unreported (datasheet A09420); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat retrieval: Citrate pH 6.0 (datasheet A09420); 20 min, 95–100 °C (standard)
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-MYH1, 1:50-1:200 (datasheet A09420)
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	MYH1-positive staining in cardiomyocytes of heart muscle (HPA tissue IHC: High). HPA tissue profile: Selective cytoplasmic expression in heart and skeletal muscle. No signal in the no-primary control.

💡Decision noteStart with heat-mediated citrate retrieval at pH 6.0 for the catalog antibody; the rabbit study also used pH 6 retrieval for MYH1 (datasheet A09420; PMC12331014).

Section 2

What Is the Expected MYH1 Staining Pattern?

MYH1 should appear in the cytoplasm of muscle cells, consistent with its location in myofibrillar thick filaments and its lack of a transmembrane segment (UniProt P12882 localisation and topology). HPA reports high staining in skeletal myocytes and cardiomyocytes, with selective cytoplasmic expression in skeletal and heart muscle (HPA tissue IHC). Interpret that pattern with caution: HPA rates its tissue profile Enhanced but notes medium agreement with RNA data and antibody recognition of proteins from multiple genes (HPA tissue IHC).

What am I looking at on my slide?
Strong cytoplasmic staining in skeletal myocytes, with myofibrillar distribution where structure is visible (HPA: High in skeletal myocytes; UniProt P12882 localisation).	This fits the expected positive pattern. Score signal within muscle cells rather than staining across the section; MYH1 is associated with myofibrillar thick filaments (UniProt P12882 localisation). HPA staining alone cannot establish that every positive fibre expresses MYH1 specifically because its profile carries a multiple-gene warning (HPA tissue IHC).
Strong cytoplasmic staining in cardiomyocytes (HPA: High in cardiomyocytes).	This matches the supplied HPA tissue observation, although HPA also describes skeletal muscle as RNA enriched (HPA tissue IHC). Treat heart staining as an observed antibody pattern, not independent proof of MYH1-specific protein abundance: HPA reports medium RNA agreement and possible recognition of proteins from multiple genes (HPA tissue IHC).
Predominantly nuclear, surface-rim or extracellular staining instead of muscle-cell cytoplasm.	That distribution conflicts with the documented myofibrillar cytoplasmic location and absence of a transmembrane segment (UniProt P12882 localisation and topology). First assess staining artefact and antibody specificity; the compartment mismatch alone does not identify its cause (general IHC interpretation).
Conspicuous staining in adipocytes, respiratory epithelial cells or marrow hematopoietic cells.	HPA reports these sampled cell types as not detected (HPA tissue IHC). A positive result there raises concern for cross-reactivity or endogenous detection activity; compare its location and intensity with a known-positive muscle section and the relevant detection controls (general IHC practice).
Diffuse section-wide colour, or no muscle-cell signal in a known-positive skeletal muscle section.	Diffuse colour makes cell-specific scoring unreliable; absent signal contradicts the reported high skeletal-myocyte staining (HPA tissue IHC). Use controls to distinguish a detection/background problem from a failed stain, then review routine IHC processing steps without assuming a MYH1-specific fixation effect (general IHC practice).

💡Expected MYH1 appearanceCall a section positive when skeletal myocytes show strong cytoplasmic, preferably myofibrillar staining; high cardiomyocyte staining is also reported (HPA tissue IHC; UniProt P12882 localisation). Predominantly nuclear or diffuse staining is discordant with that pattern (UniProt P12882 localisation; general IHC interpretation).

How each factor affects the staining
Tissue and cell context (HPA tissue IHC).	HPA reports high staining in skeletal myocytes and cardiomyocytes, while its listed adipocytes, respiratory epithelial cells and marrow hematopoietic cells are not detected (HPA tissue IHC). Use these observations to choose comparative sections, while retaining HPA's medium RNA agreement and multiple-gene caveat (HPA tissue IHC).
Antibody interpretation (HPA antibody validation; HPA tissue IHC).	The listed IHC ratings differ: HPA001349 is Supported and CAB010759 is Enhanced (HPA antibodies). An Enhanced rating supports reproducibility or orthogonal agreement under HPA's criteria; it does not remove the tissue profile's warning that staining may represent proteins from more than one gene (HPA tissue IHC).
Intracellular target location (UniProt P12882).	MYH1 is assigned to cytoplasmic myofibrillar thick filaments and has no transmembrane segment (UniProt P12882 localisation and topology). These annotations support judging muscle-cell cytoplasm as the relevant compartment; they do not supply a target-specific antigen retrieval condition or fixation sensitivity (UniProt P12882 record).
Q: Should focal-adhesion IF signal define the IHC positive pattern? (HPA subcellular ICC-IF).	A: No. HPA labels focal adhesion sites uncertain and warns that the ICC-IF antibodies target proteins from multiple genes (HPA subcellular ICC-IF). Keep that observation qualified on the separate IF/ICC page; judge paraffin IHC against the reported muscle-cell cytoplasmic pattern (HPA tissue IHC).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
No signal in skeletal myocytes despite an intact known-positive section (HPA: High in skeletal myocytes).	A failed staining run or insufficient detection is possible; the supplied sources do not establish MYH1-specific fixation sensitivity (general IHC practice; UniProt P12882 record).	Check the positive control, antibody application, detection reagents and counterstain. Review the selected IHC antibody's validated procedure for antigen retrieval before changing conditions; do not infer a MYH1-specific retrieval requirement from HPA staining levels (general IHC practice).
Muscle cells stain mainly in nuclei or along cell borders (UniProt P12882 localisation and topology).	The compartment differs from the documented cytoplasmic myofibrillar location; staining artefact or cross-reactivity is possible, but location alone cannot distinguish them (UniProt P12882 localisation; general IHC interpretation).	Compare with a known-positive muscle section, inspect morphology and run an appropriate primary-omission control. Interpret persistent compartment mismatch cautiously because the HPA tissue profile warns of multiple-gene recognition (general IHC practice; HPA tissue IHC).
Adipocytes or respiratory epithelial cells appear positive (HPA: Not detected in those cells).	Cross-reactivity or endogenous chromogenic detection activity may account for unexpected staining; HPA's multiple-gene warning adds a specificity concern (general IHC practice; HPA tissue IHC).	Compare the signal with muscle-cell cytoplasm and check primary-omission and detection controls. Address endogenous enzyme activity using the detection system's standard controls before assigning unexpected cells MYH1 expression (general IHC practice).
Diffuse brown background obscures myocytes (general chromogenic IHC observation).	Nonspecific reagent binding, inadequate washing or endogenous detection activity can create background in chromogenic IHC (general IHC practice).	Check primary-omission and reagent controls, then review blocking, washes and antibody concentration within the chosen antibody's IHC procedure. Score only distinguishable cellular staining; HPA's high muscle staining does not validate diffuse colour (general IHC practice; HPA tissue IHC).
Heart staining seems stronger than expected from skeletal muscle RNA enrichment (HPA tissue IHC).	HPA reports high cardiomyocyte staining alongside skeletal-muscle-enriched RNA, medium RNA agreement and possible recognition of proteins from multiple genes (HPA tissue IHC).	Record heart staining as an observed IHC result and compare cell location and controls across sections. Avoid converting relative colour intensity into a MYH1 expression ranking without independent specificity evidence (HPA tissue IHC; general IHC interpretation).
Two antibodies give different muscle or off-target patterns (HPA antibody validation; HPA tissue IHC).	The listed IHC validation levels differ, and the HPA tissue profile cautions that antibodies can recognize proteins from multiple genes (HPA antibodies; HPA tissue IHC).	Record antibody identity and IHC validation status, compare matched controls and the cytoplasmic muscle-cell pattern, and report unresolved disagreement. Neither a matching location nor an Enhanced rating alone proves MYH1-specific staining (HPA antibodies; HPA tissue IHC; UniProt P12882 localisation).

Source: UniProt P12882 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing MYH1 staining

Immunohistochemistry of paraffin-embedded Human skeletal muscle using MYH1 Rabbit pAb at dilution of 1:50 .Perform high pressure antigen retrieval with 10 mM citrate buffer pH 6.0 before commencing with IHC staining protocol.

A09420 · IHCImmunohistochemistry of paraffin-embedded Human skeletal muscle using MYH1 Rabbit pAb at dilution of 1:50 .Perform high pressure antigen ret

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000109061-MYH1/tissue/cerebral+cortex#imid_2734480

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000109061-MYH1/tissue/colon#imid_2734406

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000109061-MYH1/tissue/liver#imid_2734391

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000109061-MYH1/tissue/kidney#imid_2734408

HPA · ICC-IF OE19Human Protein Atlas immunofluorescence, OE19, antibody HPA001349; green = MYH1, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for MYH1 IHC & IF

🧪Run skeletal muscle first; myocytes should show myofibrillar staining (HPA: High in skeletal-muscle myocytes; UniProt P12882: thick filaments of myofibrils). Use adipose tissue as the negative tissue (HPA: Not detected in adipocytes), and assess nonmyocyte areas on the skeletal-muscle slide for absence of specific myofibrillar staining (standard IHC practice).

Positive control tissue: Heart muscle (Cardiomyocytes, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show MYH1 in OE19, SuSa, U2OS, with annotated localisation: Focal adhesion sites (uncertain) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control, an isotype-matched rabbit polyclonal control, and MYH1 knockout tissue if available (selected tissue-IHC caption: rabbit pAb; standard IHC practice). For chromogenic IHC, block endogenous peroxidase and check endogenous biotin if using biotin-based detection; for IF, assess skeletal-muscle autofluorescence with the no-primary control (standard IHC/IF practice).

⚠️Feasibility: No supplied source reports a MYH1-specific fixation window or fixation effect, and the selected A09420 paraffin-section caption leaves the fixative unreported (selected tissue-IHC caption). That caption specifies high-pressure retrieval in 10 mM citrate buffer at pH 6.0 and a 1:50 antibody dilution, but does not establish whether retrieval is required under other conditions (selected tissue-IHC caption). The supplied evidence does not establish whether frozen sections or IF are easier; skeletal-muscle autofluorescence may complicate IF interpretation (standard IF practice).

HPA tissue IHC evidence for MYH1

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — 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	High	Protein (IHC)	HPA →
Skeletal muscle	Myocytes	High	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 MYH1 IHC Tips

Troubleshoot MYH1 staining in paraffin sections by checking retrieval, cytoplasmic localization, antibody specificity, and matched controls.

What retrieval should I try first for weak MYH1 staining in paraffin sections?

Start with heat-mediated, high-pressure retrieval in 10 mM citrate buffer at pH 6.0 for paraffin-section MYH1 IHC (datasheet A09420). The selected skeletal-muscle image used this retrieval and a 1:50 antibody dilution, so use it as the initial comparison condition (A09420 tissue-IHC caption). If staining remains weak, compare a modestly longer heating interval with the original condition while keeping section thickness, cooling, and detection consistent (general IHC practice). Judge improvement by cytoplasmic staining in myocytes and preservation of tissue morphology, rather than by stronger staining at section edges (UniProt P12882 localization; HPA tissue IHC; general IHC practice).

How should I troubleshoot MYH1 staining when fixation details are incomplete?

Target-specific sensitivity to fixation is unknown, and the selected paraffin-section caption does not state a fixative (A09420 tissue-IHC caption). Record the fixative and fixation duration for each specimen, then compare sections processed with the same retrieval and detection conditions (general IHC practice). If a newly processed batch stains weakly, compare it with a previously successful section in the same run before changing the antibody dilution or retrieval time (general IHC practice). Treat any difference as a processing observation until matched specimens establish its cause; neither the reported tissue pattern nor MYH1 topology establishes a fixation effect (HPA tissue IHC; UniProt P12882 topology).

Which staining compartment and cells support a plausible MYH1 IHC result?

Look for cytoplasmic staining associated with myofibrils in myocytes, consistent with MYH1 in thick filaments and with high staining reported in skeletal-muscle myocytes (UniProt P12882 localization; HPA tissue IHC). High staining is also reported in heart-muscle cardiomyocytes, although the HPA antibody may recognize proteins from more than one gene (HPA tissue IHC). Predominantly nuclear staining or a crisp cell-membrane outline warrants investigation because MYH1 has no transmembrane segment and is assigned to myofibrils (UniProt P12882 topology and localization). Compare suspicious patterns with an omission control and adjacent intact tissue before calling them positive (general IHC practice).

How can I assess whether the antibody distinguishes MYH1 from related myosins?

The record lists 0 annotated MYH1 isoforms, but that does not establish antibody specificity among related myosin proteins (UniProt P12882 isoforms; HPA tissue-IHC reliability note). Ask for the antibody's immunogen or mapped epitope, then check whether that sequence is shared with related myosins before assigning staining specifically to MYH1 (general antibody-validation practice). MYH1 contains an SH3-like region at residues 33–82, a motor region at 86–782, and an IQ region at 785–814; the supplied caption does not identify which region the antibody recognizes (UniProt P12882 domains; A09420 tissue-IHC caption). Interpret staining alongside an independently validated specificity control, especially because HPA flags recognition of proteins from multiple genes (HPA tissue-IHC reliability note; general IHC practice).

How should I plan a MYH1 immunofluorescence comparison alongside this IHC assay?

Use the paraffin-section IHC result as a comparison, while validating antibody staining separately under the chosen IF conditions (A09420 tissue-IHC caption; general IF practice). Multiplex with a marker that identifies myocytes, the expected positive cell type, and choose spectrally separated fluorophores with a far-red channel where tissue autofluorescence interferes (HPA tissue IHC; general IF practice). MYH1 is cytoplasmic and myofibrillar, with no transmembrane segment, so permeabilize fixed cells sufficiently for antibody access to that intracellular compartment (UniProt P12882 localization and topology; general IF practice). Include single-color and secondary-only controls to distinguish channel bleed-through and background from MYH1 signal (general IF practice).

What controls help distinguish diffuse MYH1 signal from chromogenic background?

Compare the MYH1 slide with a primary-antibody omission control processed through the same chromogenic detection steps (general IHC practice). Apply a peroxidase block before a peroxidase-based DAB reaction, and check whether pigment or endogenous enzyme activity remains visible without primary antibody (general IHC practice). If diffuse color persists only with primary antibody, reassess the 1:50 starting dilution, blocking, washes, and retrieval while preserving a matched positive section (A09420 tissue-IHC caption; general IHC practice). Give greatest weight to cytoplasmic staining in intact myocytes, the compartment and cell type supported by the supplied localization evidence (UniProt P12882 localization; HPA tissue IHC).

How should I quantify MYH1 chromogenic staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define intact myocytes as the scoring population and exclude folds, torn edges, and necrotic regions before measuring staining (HPA tissue IHC; general IHC practice). Record the percentage of positive myocytes and their cytoplasmic intensity on a 0–3 scale; an H-score sums each intensity multiplied by its cell percentage, giving 0–300 (general IHC scoring practice). Normalize positive-cell counts to all evaluable myocytes, or normalize stained area to evaluable muscle area, using the same rule for every section (general IHC scoring practice). Keep retrieval, detection, counterstain, imaging, and threshold settings consistent, and report the HPA cross-gene specificity caveat when interpreting MYH1-specific scores (general IHC practice; HPA tissue-IHC reliability note).

When is apparent MYH1 staining likely to be artefactual?

A plausible result places chromogenic signal in myocyte cytoplasm, consistent with MYH1 in myofibrillar thick filaments and with the reported skeletal-muscle staining pattern (UniProt P12882 localization; HPA tissue IHC). Treat isolated nuclear or membrane-rim staining cautiously because it conflicts with the supplied MYH1 localization and absence of a transmembrane segment (UniProt P12882 localization and topology). Compare strong edge staining, necrotic areas, and signal in the primary-antibody omission control with intact central tissue to assess processing or endogenous-enzyme artefacts (general IHC practice). Even anatomically plausible staining is not definitive proof of MYH1 alone: HPA cautions that its staining antibody targets proteins from more than one gene (HPA tissue-IHC reliability note).

Boster reagents

Best MYH1 / Myosin-1 IHC Antibodies

A09420 has a human skeletal muscle paraffin-section IHC image (catalog IHC caption); IF/ICC is listed for the antibody, with human, mouse and rat reactivity (catalog applications and reactivity).

Real IHC data Immunohistochemistry of paraffin-embedded Human skeletal muscle using MYH1 Rabbit pAb at dilution of 1:50 .Perform high pressure antigen retrieval with 10 mM citrate buffer pH 6.0 before commencing with IHC staining protocol.

Anti-Myosin-1 MYH1 Antibody

Cat # A09420

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A09420 will render with an IHC image of human skeletal muscle paraffin sections stained at 1:50 after citrate pH 6.0 retrieval (catalog IHC caption). IF/ICC is listed, and human, mouse and rat reactivity is reported; no IF image is supplied (catalog applications, reactivity and image records).

Which to pick: Choose A09420 for paraffin-section IHC: its own image documents human skeletal muscle staining, and the listed IHC dilution range is 1:50–1:200 (catalog IHC caption and dilution). A09420 is also the listed IF/ICC option, with a 1:50–1:100 dilution range, and the listed cross-species option for human, mouse and rat; it is polyclonal (catalog applications, dilution, reactivity and antibody details). The IHC caption specifies paraffin embedding and citrate retrieval but does not report the fixative (catalog IHC caption).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry P12882 (MYH1_HUMAN, Myosin-1).
Human Protein Atlas. MYH1 tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. MYH1 subcellular location (ICC-IF): Localized to the focal adhesion sites. Caution: Based on antibodies targeting proteins from multiple genes..
Human Protein Atlas. MYH1 antibody validation summary (2 antibodies).
Local tissue response to a C-X-C motif chemokine ligand 12 therapy for fecal incontinence in a rabbit model. American journal of physiology. Gastrointestinal and liver physiology 2025 — PMC12331014.
Metformin enhances external urethral sphincter integrity and restores continence via AMPK activation in a rat model of stress urinary incontinence. Animal models and experimental medicine 2026 — PMC13242725.
Long noncoding RNA SMUL suppresses SMURF2 production-mediated muscle atrophy via nonsense-mediated mRNA decay. Molecular therapy. Nucleic acids 2021 — PMC7807096.
LncRNA SMARCD3-OT1 Promotes Muscle Hypertrophy and Fast-Twitch Fiber Transformation via Enhancing SMARCD3X4 Expression. International journal of molecular sciences 2022 — PMC9105468.
PubMed PMID:10388558 — UniProt-cited evidence.
PubMed PMID:16625196 — UniProt-cited evidence.
PubMed PMID:15489334 — UniProt-cited evidence.