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EYA4 / Protein phosphatase EYA4 · IHC design guide
Design Immunohistochemistry for EYA4
Plan EYA4 IHC in paraffin sections using heart cardiomyocytes and skeletal myocytes as positive tissue controls (HPA tissue IHC). The catalog antibody is listed for IHC at 0.5–1 μg/mL (datasheet A04516-3); assess cytoplasmic and nuclear staining (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
EYA4 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 and nuclear staining in most tissues (HPA tissue IHC) | |
| Staining pattern | High in cardiomyocytes and myocytes; cytoplasmic/nuclear (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A04516-3) | |
| Positive control | Heart muscle+4 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 conditions consistent across samples (standard IHC practice; not target-specific) | |
| Caveat | Staining has medium consistency with RNA expression (HPA tissue IHC) | |
| Regulation | Higher expression in heart and skeletal muscle (UniProt) | |
| Isoform / epitope | 5 isoforms; epitope coverage is unreported (UniProt) |
Section 1
Recommended EYA4 IHC & IF Protocols
Start with the catalog antibody’s IHC-P protocol (datasheet: EDTA pH 8.0 retrieval). The published EYA4 protocols below cover liver, bile duct, gastric cardia and breast specimens (cited PMC articles).
| Sample | Paraffin-embedded human mammary cancer tissue; fixative not specified (datasheet A04516-3) |
| Fixation | Image fixative and duration unreported (datasheet A04516-3); 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 A04516-3); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A04516-3) |
| Primary antibody | Rabbit anti-EYA4, 0.5-1μg/ml (datasheet A04516-3) |
| Primary incubation | Overnight at 4 °C (datasheet A04516-3) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A04516-3) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | EYA4-positive staining in cardiomyocytes of heart muscle (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and nuclear expression in most tissues, including heart and skeletal muscle. No signal in the no-primary control. |
💡Decision noteTry heat-mediated EDTA at pH 8.0 first (datasheet: A04516-3); citrate retrieval at pH 6.0 is a published alternative for liver and gastric cardia sections (PMC6533515; PMC9843510).
Section 2
What Is the Expected EYA4 Staining Pattern?
EYA4 staining is expected in the cytoplasm and nucleus across many tissues, with strong staining in heart cardiomyocytes and skeletal myocytes (HPA: tissue IHC). EYA4 has no transmembrane segment (UniProt O95677: topology). Use this as a cell and compartment pattern, with appropriate caution: HPA rates its tissue IHC profile Enhanced but reports medium consistency between staining and RNA expression (HPA: reliability).
| Strong nuclear and cytoplasmic staining in cardiomyocytes or skeletal myocytes. | This fits the strongest supplied tissue observations (HPA: High in heart cardiomyocytes and skeletal myocytes) and EYA4's reported compartments (UniProt O95677: cytoplasm; nucleus). Judge staining in the named cells, rather than treating every structure in the section as a positive control. |
| Predominantly membrane-rim staining, with little intracellular signal. | Question target specificity: a membrane-only pattern conflicts with the reported cytoplasmic and nuclear distribution (HPA: tissue IHC; UniProt O95677: subcellular location) and the absence of a transmembrane segment (UniProt O95677: topology). Check the staining pattern before assigning it to EYA4. |
| Strong staining in adipocytes, germinal center cells, or smooth muscle cells. | These named cell populations were not detected in the supplied tissue profile (HPA: adipose tissue; lymph node; smooth muscle). Unexpected staining warrants checks for cross-reactivity or endogenous detection activity (standard IHC practice); it does not, by itself, establish either cause. |
| Diffuse color across cells and surrounding tissue, obscuring cell boundaries. | The result cannot be scored confidently for EYA4's intracellular pattern (HPA: tissue IHC; UniProt O95677: subcellular location). Diffuse background can arise from nonspecific binding or detection chemistry (standard IHC practice); compare controls before interpreting weak apparent positives. |
| No staining in cardiomyocytes or skeletal myocytes. | These are the supplied High-staining cell populations (HPA: heart muscle; skeletal muscle). First check whether the run produced a usable positive-control signal (standard IHC practice). A negative section alone cannot distinguish absent antigen from a staining failure. |
💡Expected EYA4 appearanceCall a result positive when cardiomyocytes or skeletal myocytes show strong intracellular nuclear and cytoplasmic staining (HPA: High in both; HPA: tissue IHC); diffuse background or isolated membrane-rim color does not match that pattern (HPA: tissue IHC; UniProt O95677: topology).
| Which tissue and cell provide the clearest reference? | Heart cardiomyocytes and skeletal myocytes are High by IHC (HPA: tissue IHC), consistent with high expression in heart and skeletal muscle (UniProt O95677: tissue specificity). Medium staining is also reported in selected glandular, epithelial, and neuronal cells (HPA: tissue IHC). |
| How should nuclear versus cytoplasmic signal be read? | Both compartments belong to the reported tissue pattern (HPA: tissue IHC; UniProt O95677: subcellular location). SIX3 interaction can move EYA4 from cytoplasm to nucleus (UniProt O95677: subunit); this record does not establish a tissue-specific nuclear-to-cytoplasmic ratio. |
| How strong is the pattern evidence? | HPA calls the tissue profile Enhanced while describing medium staining-to-RNA consistency (HPA: reliability). Antibody validation also differs: HPA038771 is IHC Enhanced, whereas HPA004805 and HPA038772 are IHC Approved (HPA: antibodies). Match conclusions to the antibody used. |
| Could isoforms affect interpretation? | Five EYA4 isoforms are listed (UniProt O95677: isoforms). The supplied record gives no antibody epitope or isoform coverage, so a staining difference cannot be assigned to a particular isoform from this evidence; check the assay documentation before making that claim. |
| What should ICC-IF show? | Nucleoplasmic localization is approved in the supplied ICC-IF record, with images listed for HeLa, Rh30, and U2OS (HPA: subcellular ICC-IF). This is a localization cross-check; tissue IHC also reports cytoplasmic staining (HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Heart or skeletal-muscle positive control has no signal. | The result conflicts with High staining in the named cells (HPA: tissue IHC); the cause is unresolved. | Check section quality, antibody identity and dilution, retrieval, and detection with run controls (standard IHC practice). Do not infer EYA4 absence until the positive control works. |
| Only a membrane outline stains. | A membrane-only pattern is discordant with reported intracellular staining and no transmembrane segment (HPA: tissue IHC; UniProt O95677: topology). | Review cell boundaries and the negative reagent control, then compare an independently validated antibody if available (standard IHC practice; HPA: antibody validation). |
| Adipocytes or smooth muscle cells stain strongly. | Those specific cell types are Not detected in the supplied profile (HPA: adipose tissue; smooth muscle). Cross-reactivity or endogenous detection activity is possible (standard IHC practice). | Inspect reagent controls and the staining compartment; repeat with an independently validated antibody if available (standard IHC practice; HPA: antibody validation). |
| Weak color covers the whole section. | Diffuse background obscures the expected cell and compartment distribution (HPA: tissue IHC); nonspecific binding or detection background may contribute (standard IHC practice). | Compare negative reagent controls, then optimize blocking, antibody dilution, and detection conditions one at a time (standard IHC practice). Score only interpretable cellular staining. |
| A negative-reference cell population appears faintly stained. | HPA reports Not detected for particular cells, not necessarily for every cell in the same tissue (HPA: tissue IHC). Faint color may also be background (standard IHC practice). | Identify the stained cell type first; compare its intensity and compartment with the High-staining control and negative reagent control (HPA: tissue IHC; standard IHC practice). |
| Nuclear and cytoplasmic staining vary between specimens. | Both compartments are reported for tissue IHC, and SIX3 can promote nuclear translocation (HPA: tissue IHC; UniProt O95677: subunit). The supplied evidence does not explain a particular specimen's ratio. | Record nuclear and cytoplasmic scores separately and compare matched cell types under the same run conditions (standard IHC practice); avoid assigning the difference to fixation or an isoform without further evidence. |
Experiment evidence
IHC & IF images showing EYA4 staining
A04516-3 · IHCIHC analysis of EYA4 using anti-EYA4 antibody (A04516-3). EYA4 was detected in paraffin-embedded section of human mammary cancer tissue. Hea
A04516-3 · IHCIHC analysis of EYA4 using anti-EYA4 antibody (A04516-3). EYA4 was detected in paraffin-embedded section of human rectal cancer tissue. Heat
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000112319-EYA4/tissue/cerebral+cortex#imid_10087500
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000112319-EYA4/tissue/colon#imid_10087426
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000112319-EYA4/tissue/liver#imid_10087427
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000112319-EYA4/tissue/kidney#imid_10087444
HPA · ICC-IF HeLaHuman Protein Atlas immunofluorescence, HeLa, antibody HPA004805; green = EYA4, blue = nucleus, red = microtubules.
HPA · ICC-IF HeLaHuman Protein Atlas immunofluorescence, HeLa, antibody HPA004805; green = EYA4, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for EYA4 IHC & IF
🧪Run heart muscle first: cardiomyocytes should stain for EYA4 (HPA: High in cardiomyocytes). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes); on the heart slide, treat non-cardiomyocyte cells as internal negatives only if they remain at background, because their EYA4 level is not specified (HPA: High in cardiomyocytes).
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 EYA4 in HeLa, Rh30, U2OS, with annotated localisation: Nucleoplasm (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; a rabbit IgG isotype control matched to the primary antibody’s format and concentration; and EYA4-knockout tissue or cells as a biological negative (selected-SKU caption: rabbit anti-EYA4 antibody; standard IHC controls). Check endogenous peroxidase and endogenous biotin in heart muscle when using the caption’s biotin–streptavidin/DAB detection method (selected-SKU caption: SABC with DAB; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the fixative in the selected A04516-3 paraffin-section caption is unreported (selected-SKU caption: fixative not stated). The caption uses heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required under other conditions (selected-SKU caption: EDTA retrieval). Is frozen-section IHC or IF/ICC easier? The supplied evidence does not establish that comparison; ICC-IF images show nucleoplasmic EYA4 in HeLa, Rh30 and U2OS cells, while heart muscle requires attention to peroxidase and biotin background with the caption’s chromogenic method (HPA: Nucleoplasm approved; HPA: ICC-IF image cell lines; selected-SKU caption: SABC with DAB; standard IHC practice).
HPA tissue IHC evidence for EYA4
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 |
|---|---|---|---|---|
| Heart muscle | Cardiomyocytes | High | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | Not detected | Protein (IHC) | HPA → |
| Oral mucosa | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced EYA4 IHC Tips
Troubleshoot EYA4 staining in paraffin sections by checking retrieval, compartment, cell type and controls before comparing staining scores.
What retrieval should I try first when EYA4 staining is weak?
Start with heat-mediated EDTA retrieval at pH 8.0 for paraffin sections (datasheet A04516-3). The selected tissue image used that retrieval before overnight incubation at 4°C with 1 µg/mL antibody, so reproduce those conditions when assessing weak staining (caption A04516-3). Keep heating and cooling consistent across slides, since uneven retrieval can produce misleading differences within a batch (standard IHC practice). If staining remains weak, adjust heating time on matched sections while keeping buffer and detection conditions fixed; assess tissue integrity alongside signal (standard IHC practice). Compare the result with cardiomyocytes or skeletal myocytes, where staining is reported as high (HPA: High in cardiomyocytes and skeletal myocytes).
Could fixation explain weak or uneven EYA4 staining?
The selected paraffin-section caption does not state a fixative, and target-specific fixation sensitivity is unknown (caption A04516-3). Record the fixative, fixation duration and processing history for each specimen before attributing a staining difference to EYA4 expression (standard IHC practice). Compare sections with similar handling and include a positive-control tissue processed alongside them; heart cardiomyocytes and skeletal myocytes have reported high staining (HPA: High in cardiomyocytes and skeletal myocytes). If signal varies across a block, inspect morphology and compare sections from its interior and edge before changing antibody concentration (standard IHC practice). Evaluate any fixation change with matched specimens, because the supplied evidence does not establish an EYA4-specific fixation effect (caption A04516-3).
Should EYA4 appear in nuclei, cytoplasm or both?
Score nuclear and cytoplasmic staining separately: EYA4 is annotated in both compartments, while tissue IHC reports both across most tissues (UniProt O95677 subcellular location; HPA tissue IHC). Nucleoplasmic localisation is also reported from ICC/IF images, providing a useful compartment check rather than an IHC intensity standard (HPA subcellular). SIX3 interaction can move EYA4 from cytoplasm to nucleus, so a shift in compartment may have biological context (UniProt O95677 subunit). Confirm nuclear signal against a counterstain and inspect cell boundaries before assigning mixed staining (standard IHC practice). Treat diffuse extracellular deposit or staining confined to damaged tissue as suspect and compare it with a matched negative control (standard IHC practice).
How should I interpret staining when the antibody epitope is unspecified?
EYA4 has 5 annotated isoforms, and the supplied caption does not identify the antibody epitope (UniProt O95677 isoforms; caption A04516-3). Consequently, this IHC signal cannot establish which isoform is present without epitope information or an independently validated isoform-specific assay (UniProt O95677 isoforms; standard IHC interpretation). EYA4 has no annotated transmembrane segment, while acetylation at residue 1 and phosphorylation at residue 361 are recorded modifications (UniProt O95677 topology and modified residues). Do not infer that either modification changes antibody binding without an epitope map and direct testing (standard IHC interpretation). When comparing specimens, use the same antibody and retrieval conditions, and describe the result as EYA4 immunoreactivity rather than isoform abundance (standard IHC practice).
How can I cross-check an EYA4 IHC pattern by multiplex IF?
Use IF as a separate cross-check and assess whether EYA4 overlaps a marker for the expected cell type, such as cardiomyocytes or skeletal myocytes in their respective tissues (HPA: High in cardiomyocytes and skeletal myocytes; standard IF practice). Choose fluorophores after examining unstained tissue autofluorescence, and include single-label controls to assess bleed-through (standard IF practice). Permeabilise for intracellular epitopes, then check nuclear and cytoplasmic signal separately because EYA4 is annotated in both compartments and has no transmembrane segment (UniProt O95677 subcellular location and topology; standard IF practice). The supplied product image documents chromogenic paraffin-section IHC, with no fixative stated, so determine IF fixation and permeabilisation empirically for the IF specimens (caption A04516-3; standard IF practice).
How do I reduce diffuse brown background without losing EYA4 signal?
First inspect a section without primary antibody to locate detection-system background, then compare it with the EYA4-stained section (standard IHC practice). The selected image used 10% goat serum blocking, a biotinylated secondary antibody, a streptavidin–biotin complex and DAB, so check nonspecific secondary binding and endogenous biotin when reproducing it (caption A04516-3; standard IHC practice). Include a peroxidase block and inspect the no-primary control for residual DAB deposition (standard chromogenic IHC practice). Titrate the primary antibody around the documented 1 µg/mL condition while holding retrieval and development constant (caption A04516-3; standard IHC practice). Preserve the expected cellular pattern when choosing a lower-background condition (HPA tissue IHC; standard IHC practice).
What should I measure when comparing EYA4 across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because EYA4 staining is reported in cytoplasm and nuclei (HPA tissue IHC; UniProt O95677 subcellular location). For each compartment, record the percentage of positive cells and staining intensity, or calculate an H-score using prespecified intensity bins (standard IHC practice). Normalise counts to the number of evaluable cells in that population; if measuring positive-cell density, report it per mm² of evaluable tissue (standard IHC practice). Exclude folds, necrosis and section edges by a consistent rule, and keep retrieval and DAB development comparable between slides (standard IHC practice). Report cell-type-specific scores because cardiomyocytes and skeletal myocytes have high reported staining whereas adipocytes are not detected (HPA tissue IHC).
Which EYA4 staining patterns warrant suspicion before I call a section positive?
A convincing result has cellular nuclear or cytoplasmic staining in an appropriate population, consistent with EYA4 localisation and its reported tissue IHC profile (UniProt O95677 subcellular location; HPA tissue IHC). Strong cardiomyocyte or skeletal-myocyte signal is plausible, while adipocyte staining needs closer control review because those cells are reported as not detected (HPA tissue IHC). Treat staining concentrated at section edges, folds or necrotic areas as a processing artefact until repeat sections support it (standard IHC practice). Check no-primary and peroxidase-block controls when brown deposit could reflect endogenous enzyme activity or the detection system (standard chromogenic IHC practice). Interpret discrepancies cautiously: the HPA tissue profile has Enhanced reliability but only medium consistency between staining and RNA expression (HPA tissue IHC).
Boster reagents
Best EYA4 / Protein phosphatase EYA4 IHC Antibodies
A04516-3 has real IHC data from paraffin sections of human mammary cancer tissue (IHC image caption). The catalog lists human, monkey and rat reactivity (catalog reactivity); no IF image is supplied (catalog images).
Real IHC data
Anti-EYA4 Antibody ®
Cat # A04516-3
A04516-3 will render with its human mammary cancer tissue IHC figure (IHC image caption). Its other supplied IHC caption shows human rectal cancer tissue; the catalog lists IHC among its applications and human, monkey and rat reactivity (IHC image caption; catalog applications and reactivity).
Which to pick: Choose A04516-3 for human paraffin-section IHC: its figure documents EDTA retrieval at pH 8.0 and primary antibody at 1 μg/ml (IHC image caption); the fixative is unreported (IHC image caption). No IF/ICC application or image is supplied, so there is no validated IF/ICC choice here (catalog applications and images). A04516-3 lists monkey and rat reactivity, but the supplied IHC images show human tissue only; clonality is unreported (catalog reactivity; IHC image captions; catalog clone field).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O95677 (EYA4_HUMAN, Protein phosphatase EYA4).
- Human Protein Atlas. EYA4 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. EYA4 subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. EYA4 antibody validation summary (3 antibodies).
- EYA4 serves as a prognostic biomarker in hepatocellular carcinoma and suppresses tumour angiogenesis and metastasis. Journal of cellular and molecular medicine 2019 — PMC6533515.
- EYA4 gene functions as a prognostic marker and inhibits the growth of intrahepatic cholangiocarcinoma. Chinese journal of cancer 2016 — PMC4966169.
- Aberrant DNA methylation and expression of EYA4 in gastric cardia intestinal metaplasia. Saudi journal of gastroenterology : official journal of the Saudi Gastroenterology Association 2022 — PMC9843510.
- EYA4 promotes breast cancer progression and metastasis through its role in replication stress avoidance. Molecular cancer 2023 — PMC10543271.
- PubMed PMID:9887327 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:14574404 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for EYA4 (Protein phosphatase EYA4). Expected localisation Cytoplasmic and nuclear staining in most tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A04516-3). Validated IHC antibody: A04516-3 at 0.5-1μg/ml (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 human mammary cancer tissue; fixative not specified (datasheet A04516-3); 2. Fixation: Image fixative and duration unreported (datasheet A04516-3); 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 A04516-3); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A04516-3); 8. Primary antibody: Rabbit anti-EYA4, 0.5-1μg/ml (datasheet A04516-3); 9. Primary incubation: Overnight at 4 °C (datasheet A04516-3). Watch for: Fixation: Keep fixation conditions consistent across samples (standard IHC practice; not target-specific); Caveat: Staining has medium consistency with RNA expression (HPA tissue IHC); Isoform / epitope: 5 isoforms; epitope coverage is unreported (UniProt); Regulation: Higher expression in heart and skeletal muscle (UniProt). Evidence: UniProt O95677, HPA tissue IHC · Enhanced, HPA ICC-IF · nucleoplasm., Datasheet A04516-3, PMC6533515, PMC4966169. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/EYA4/immunohistochemistry-ihc-guide.