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MYO5A / Unconventional myosin-Va · IHC design guide
Design Immunohistochemistry for MYO5A
Plan MYO5A chromogenic IHC in paraffin sections using the cytoplasmic CNS staining pattern reported by HPA (HPA tissue IHC). This guide covers control selection, fixation consistency, and interpretation of tissue staining.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
MYO5A 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 | Cytoplasmic in several tissues, most abundant in CNS (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic signal in cerebellar granular-layer cells and cortical neuropil (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Cerebellum+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A02216) | |
| Caveat | Parathyroid RNA is enhanced, but glandular staining is low (HPA tissue IHC) | |
| Regulation | Tissue-enhanced RNA: brain, parathyroid (HPA tissue RNA) | |
| Isoform / epitope | 3 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended MYO5A IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with one published MYO5A IHC method (PMC10740236).
| Sample | Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A02216) |
| Fixation | Image fixative and duration unreported (datasheet A02216); 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-MYO5A, 1:50-1:200 (datasheet A02216) |
| 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 | MYO5A-positive staining in cells in granular layer of cerebellum (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several tissues, most abundant in CNS. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page antigen retrieval); compare Tris/EDTA pH 9.0 if needed (PMC10740236).
Section 2
What Is the Expected MYO5A Staining Pattern?
MYO5A should appear chiefly as cytoplasmic staining in several tissues, with the strongest reported signal in the CNS (HPA tissue IHC: Enhanced; medium consistency with RNA). Look for high staining in cerebellar granular-layer cells and cerebral-cortex neuropil, and medium staining in hippocampal neurons and several glandular cell populations (HPA tissue IHC). MYO5A has no transmembrane segment (UniProt Q9Y4I1 topology).
| Cytoplasmic staining in cerebellar granular-layer cells or cerebral-cortex neuropil. | This matches the reported high-signal sites (HPA tissue IHC: High). Judge the signal against local background and the tissue architecture; neuropil staining can extend between cell bodies, so a positive result need not outline individual cells (general IHC interpretation). |
| Predominantly nuclear, membranous, or extracellular staining in an otherwise positive region. | Treat the compartment mismatch as a warning of nonspecific signal or a detection artefact (HPA tissue IHC: cytoplasmic profile; general IHC interpretation). Check whether cytoplasmic signal remains after appropriate negative controls; UniProt does not annotate a MYO5A subcellular location (UniProt Q9Y4I1). |
| Strong staining in a cell population reported as undetected. | A signal in adipocytes, cardiomyocytes, or skeletal-muscle myocytes conflicts with those HPA observations (HPA tissue IHC: Not detected in these cells). Investigate antibody cross-reactivity or endogenous chromogenic activity before assigning it to MYO5A (general IHC practice). |
| Uniform color across tissue, lumina, and empty areas. | Diffuse staining that ignores cell boundaries is difficult to score as MYO5A (general IHC interpretation). In chromogenic IHC, inadequate blocking, excess primary antibody, or residual endogenous enzyme activity can contribute to background; compare a matched negative control (general IHC practice). |
| No signal in a cerebellar positive-control section. | Absence in granular-layer cells conflicts with the reported high staining there (HPA tissue IHC: High). First assess tissue preservation and the run controls, then review retrieval, primary-antibody dilution, and detection performance using the antibody's validated IHC-P procedure (general IHC practice). |
💡Expected MYO5A appearanceCall a section positive when cytoplasmic signal is clear in the expected cells, especially high-signal cerebellar granular-layer cells or cortical neuropil; isolated nuclear or widespread cell-independent color is suspect (HPA tissue IHC: High, cytoplasmic profile; general IHC interpretation).
| Choice of reference tissue | Cerebellar granular-layer cells and cerebral-cortex neuropil are high-signal references; hippocampal neurons and breast, colon, endometrial, or fallopian-tube glandular cells have medium reported staining (HPA tissue IHC). A weak result in a medium-signal site alone is less informative than failure in a high-signal site (general IHC interpretation). |
| Antibody validation and interpretation limit | HPA lists HPA001356 as IHC Enhanced, meaning its pattern is supported by independent antibodies or orthogonal data (HPA antibody validation). The tissue profile still has medium consistency with RNA, so score the observed cells and compartment rather than treating every RNA-enriched tissue as a strong protein control (HPA tissue IHC). |
| Epitope and isoform coverage | MYO5A has three listed isoforms and defined motor, IQ, and Dilute regions (UniProt Q9Y4I1). The supplied evidence gives no antibody epitope or isoform coverage; staining cannot establish which isoform is present. Check the catalog antibody's documented epitope before making isoform-specific claims (general IHC interpretation). |
| IF/ICC Q&A: should puncta appear? | Yes, depending on the preparation: HPA reports approved IF/ICC localization at focal adhesion sites and centriolar satellites, with additional cytosolic and ciliary-associated sites (HPA subcellular). These cell-image observations do not require discrete puncta in chromogenic paraffin-section IHC, where HPA reports a cytoplasmic tissue profile (HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Positive-control cerebellum is blank. | The result conflicts with high granular-layer staining (HPA tissue IHC); the failing step is not identified by that observation alone. | Confirm that the control tissue and detection run worked, then check retrieval, the primary-antibody dilution, and detection reagents against the validated IHC-P procedure (general IHC practice). |
| Only glandular tissue shows weak staining. | Several glandular populations have medium reported staining, while parathyroid glandular cells are low (HPA tissue IHC). | Compare the same run with a high-signal cerebellar or cortical section before calling the antibody negative; assess matched background and tissue preservation (HPA tissue IHC; general IHC practice). |
| Color appears in reported negative cell types. | Adipocytes, cardiomyocytes, and skeletal-muscle myocytes are reported as not detected; cross-reactivity or endogenous detection activity is possible (HPA tissue IHC; general IHC practice). | Review morphology and a no-primary control. If enzyme-based color remains without primary antibody, inspect the endogenous-enzyme blocking step (general IHC practice). |
| Nuclei dominate the staining. | The pattern conflicts with the reported cytoplasmic tissue profile (HPA tissue IHC); nonspecific staining or counterstain confusion may explain it (general IHC interpretation). | Compare with a no-primary section and inspect the chromogen separately from the nuclear counterstain; avoid scoring nuclear-only signal as MYO5A (general IHC practice). |
| Diffuse background hides cellular staining. | Excess primary antibody, inadequate blocking, or residual endogenous chromogenic activity can raise background (general IHC practice). | Use matched negative controls to locate the source, then review blocking, wash steps, primary-antibody dilution, and chromogen development within the validated IHC-P workflow (general IHC practice). |
| IF/ICC puncta and IHC sections look different. | HPA describes focal-adhesion and centriolar-satellite localization in IF/ICC images, but a cytoplasmic profile in tissue IHC (HPA subcellular; HPA tissue IHC). | Interpret each preparation against its own HPA pattern and controls. Do not reject a cytoplasmic paraffin-section result solely because it lacks resolvable IF/ICC puncta (HPA tissue IHC; general IHC interpretation). |
Experiment evidence
IHC & IF images showing MYO5A staining
A02216 · IHCImmunohistochemistry (IHC) analyzes of MYO5A polyclonal antibody in paraffin-embedded human breast cancer tissue at 1:50.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000197535-MYO5A/tissue/cerebral+cortex#imid_523022
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000197535-MYO5A/tissue/colon#imid_522956
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000197535-MYO5A/tissue/liver#imid_522949
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000197535-MYO5A/tissue/kidney#imid_522958
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001356; green = MYO5A, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001356; green = MYO5A, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for MYO5A IHC & IF
🧪Run cerebellum first and look for staining in cells of the granular layer (HPA: High). Use heart muscle cardiomyocytes as a negative tissue (HPA: Not detected); on the cerebellar slide, compare adjacent unstained cells with the granular-layer signal, without assuming those cells are confirmed MYO5A-negative (HPA: High in cells in granular layer).
Positive control tissue: Cerebellum (Cells in granular layer, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show MYO5A in A-431, U-251MG, U2OS, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), Sperm, with annotated localisation: Focal adhesion sites (approved), Centriolar satellite (approved) (HPA subcellular).
Technical controls: Include no-primary or secondary-only and host- and clonality-matched isotype controls, plus a MYO5A knockout sample or validated peptide-block control (standard IHC practice). For cerebellum, quench endogenous peroxidase and check vessel-associated background before scoring chromogen (standard IHC practice).
⚠️Feasibility: A MYO5A-specific fixation window and retrieval dependence are unreported in the supplied evidence; optimize antigen retrieval using the positive and negative controls (supplied target/application evidence; standard IHC practice). The A02216 paraffin-section breast-cancer caption gives 1:50 but does not report a fixative, so fixation is unconfirmed (selected IHC caption). Frozen sections have no supplied comparison, and IF/ICC is not demonstrably easier despite documented focal-adhesion and centriolar-satellite localization; check cerebellar vessel-associated peroxidase background when interpreting IHC (HPA: approved subcellular locations; standard IHC practice). The selected A02216 tissue-IHC caption documents paraffin sections, but does not specify the fixative (selected-SKU IHC image A02216).
HPA tissue IHC evidence for MYO5A
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data.). 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 |
|---|---|---|---|---|
| Cerebellum | Cells in granular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuropil | High | Protein (IHC) | HPA → |
| Appendix | Lymphoid tissue | Medium | Protein (IHC) | HPA → |
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
| Colon | Glandular cells | 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 → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced MYO5A IHC Tips
Troubleshoot MYO5A staining in paraffin section chromogenic IHC using the page retrieval conditions, cell specific controls, and cautious interpretation of cytoplasmic signal.
How should I adjust retrieval when MYO5A staining is weak in paraffin sections?
Begin with citrate pH 6.0 heat induced epitope retrieval at 95–98 °C for 20 min (page IHC protocol). Keep cooling, wash steps, antibody dilution, and detection constant while comparing retrieval runs (standard IHC practice). If staining remains weak in a known positive section, test a second retrieval buffer or a modest time change as a fallback, alongside the original condition (standard IHC practice). Score the same cell population in both sections; cerebellar granular layer cells offer a high staining reference, while breast glandular cells show medium staining (HPA: tissue IHC). Excessive retrieval can damage morphology and make cytoplasmic signal harder to assign (standard IHC practice).
Could fixation explain weak or uneven MYO5A staining?
Target specific fixation sensitivity is unknown from the supplied evidence; the selected antibody caption reports paraffin embedded breast cancer tissue at 1:50 but gives no fixative (A02216 IHC caption). Record each specimen’s actual fixative, fixation interval, processing history, and section age before changing the staining protocol (standard IHC practice). Compare weak sections with a similarly processed positive control, keeping retrieval at citrate pH 6.0, 95–98 °C for 20 min (page IHC protocol). Patchy staining near tissue edges should prompt a check of processing and section integrity before assigning a biological difference (standard IHC practice). Neither tissue staining patterns nor protein features establish a MYO5A specific fixation effect (HPA: tissue IHC; UniProt Q9Y4I1).
Where should convincing MYO5A signal appear in tissue IHC?
Expect predominantly cytoplasmic tissue staining, with the strongest reported signals in central nervous system samples (HPA: tissue IHC profile). Cerebellar granular layer cells stain at a high level, cerebral cortex neuropil at a high level, and breast glandular cells at a medium level (HPA: tissue IHC). MYO5A is an actin based motor involved in melanosome and vesicle transport, and the sequence has no transmembrane segment (UniProt Q9Y4I1). Focal adhesion sites and centriolar satellites are approved locations in cell imaging, but routine chromogenic sections may not resolve those structures (HPA: subcellular; standard IHC practice). Treat isolated nuclear or diffuse extracellular color as suspect until controls support it (standard IHC practice).
How can I assess whether the antibody detects the MYO5A isoform I need?
MYO5A has 3 recorded isoforms, but the supplied antibody caption does not identify its epitope or establish isoform coverage (UniProt Q9Y4I1; A02216 IHC caption). Locate the antibody’s stated immunogen or epitope, then align it against each isoform before making an isoform claim (standard antibody validation practice). The protein contains a motor domain at residues 69–763, multiple IQ motifs, and a dilute domain at 1534–1810; whether any lies within this antibody’s epitope is unknown (UniProt Q9Y4I1). Phosphorylated residues are recorded at several positions, so altered staining alone cannot identify an isoform or a phosphorylation state (UniProt Q9Y4I1; standard IHC practice). Where feasible, compare staining with independent genetic or transcript evidence in matched cells (standard antibody validation practice).
How should I design a companion IF experiment to check MYO5A localisation?
Use IF as a companion localisation check while keeping this page’s protocol choices tied to tissue IHC evidence (HPA: subcellular; A02216 IHC caption). Multiplex MYO5A with a validated marker for the expected cell population, such as a neuronal marker when examining a neuronal population, and include single stain controls (HPA: hippocampal neuronal cells; standard IF practice). Choose a fluorophore in a channel with low measured tissue autofluorescence, and check spectral bleed through before interpreting overlap (standard IF practice). MYO5A lacks a transmembrane segment, so assess intracellular epitope access with a controlled permeabilisation comparison; the antibody’s epitope location is not supplied (UniProt Q9Y4I1; A02216 IHC caption). Focal adhesion and centriolar satellite patterns provide cell imaging references, not proof of the pattern in every tissue cell (HPA: subcellular).
What should I change when MYO5A IHC shows widespread brown background?
Run no primary and reagent controls to separate antibody dependent color from endogenous enzyme activity or nonspecific detection (standard IHC practice). Include an appropriate peroxidase block before chromogenic development, then compare a shorter development time while keeping the positive control visible (standard IHC practice). The selected paraffin section image used antibody dilution 1:50; treat it as an image condition, and titrate the antibody against background in your own sections (A02216 IHC caption; standard IHC practice). MYO5A participates in melanosome transport, so pigment bearing cells need particular care when judging brown signal (UniProt Q9Y4I1; standard IHC practice). Compare suspected staining with the no primary section under identical illumination and counterstain (standard IHC practice).
How can I score MYO5A IHC across heterogeneous tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, then keep segmentation and background thresholds fixed across slides (standard IHC practice). For discrete cells, report percent positive cells and an H-score based on intensity grades 0–3, giving a possible total of 0–300 (standard IHC practice). For neuropil, where individual positive cell boundaries are difficult to assign, report background corrected stained area or optical density per mm² of annotated tissue (HPA: cerebral cortex neuropil; standard IHC practice). Normalise to eligible cells or intact annotated area, and compare sections with matched processing and chromogen development (standard IHC practice). Report compartment specific results because MYO5A tissue staining is described as cytoplasmic (HPA: tissue IHC profile).
How do I distinguish true MYO5A staining from artefact?
Look for reproducible cytoplasmic signal in expected cells: cerebellar granular layer cells are reported high, while breast glandular cells are reported medium (HPA: tissue IHC). Check weak or unexpected populations against negative controls and an independently stained positive section, since the tissue IHC dataset has medium consistency with RNA expression (HPA: Enhanced reliability description; standard IHC practice). Purely nuclear color, edge concentrated staining, and signal confined to necrotic regions warrant review of morphology and controls before a positive call (HPA: tissue IHC profile; standard IHC practice). Exclude endogenous peroxidase with a no primary control and a peroxidase block, and assess whether pigment could be mistaken for chromogen (standard IHC practice). Interpret focal adhesion or centriolar patterns cautiously because those locations come from cell imaging (HPA: subcellular).
Boster reagents
Best MYO5A / Unconventional myosin-Va IHC Antibodies
The catalog covers human tissue IHC and human, mouse, and rat IF/ICC; the supplied figure documents human paraffin-section IHC (catalog applications, reactivity, and A02216 image caption).
Real IHC data
Anti-Unconventional myosin-Va MYO5A Antibody
Cat # A02216
Only A02216 renders a card; it is listed for human IHC (catalog: A02216 applications and reactivity). Its own figure shows paraffin-embedded human breast cancer tissue at 1:50, with no fixative reported (catalog: A02216 image caption).
Which to pick: Choose A02216 for human tissue IHC: this rabbit polyclonal lists a 1:50–1:200 IHC dilution, and its own figure documents a paraffin section (catalog: A02216 host, dilution, and image caption). For IF/ICC, choose A02216-1, a rabbit polyclonal listed for human, mouse, and rat at 1:50–1:100 (catalog: A02216-1 applications, reactivity, host, and dilution). For broader species reactivity in an IHC-listed reagent, A02216-2 lists human, mouse, and rat, but its paraffin-section IHC entry specifies human at 2–5 μg/ml (catalog: A02216-2 reactivity and dilution).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9Y4I1 (MYO5A_HUMAN, Unconventional myosin-Va).
- Human Protein Atlas. MYO5A tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. MYO5A subcellular location (ICC-IF): Mainly localized to the focal adhesion sites and centriolar satellites. In addition localized to the cytosol, primary cilium, basal body and calyx..
- Human Protein Atlas. MYO5A antibody validation summary (1 antibodies).
- MYO5A overexpression promotes invasion and correlates with low lymphocyte infiltration in head and neck squamous carcinoma. BMC cancer 2023 — PMC10740236.
- Gene fusions in melanocytic lesions: an updated comprehensive review. Journal of pathology and translational medicine 2026 — PMC13183473.
- Identification of three predictors of gastric cancer progression and prognosis. FEBS open bio 2020 — PMC7459413.
- PubMed PMID:9207796 — UniProt-cited evidence.
- PubMed PMID:16572171 — UniProt-cited evidence.
- PubMed PMID:8188282 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for MYO5A (Unconventional myosin-Va). Expected localisation Cytoplasmic in several tissues, most abundant in CNS (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A02216 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Cerebellum (Cells in granular layer); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A02216); 2. Fixation: Image fixative and duration unreported (datasheet A02216); 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-MYO5A, 1:50-1:200 (datasheet A02216); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A02216); Caveat: Parathyroid RNA is enhanced, but glandular staining is low (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope coverage is unspecified (UniProt); Regulation: Tissue-enhanced RNA: brain, parathyroid (HPA tissue RNA). Evidence: UniProt Q9Y4I1, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the focal adhesion sites and centriolar…, Datasheet A02216, PMC10740236. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/MYO5A/immunohistochemistry-ihc-guide.