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EPHA4 / Ephrin type-A receptor 4 · IHC design guide
Design Immunohistochemistry for EPHA4
EPHA4 IHC-P shows high staining in cerebellar molecular-layer cells and cerebral cortical neuropil (HPA tissue IHC). This guide uses those tissue patterns and the catalog antibody's 1:25 IHC-P dilution to plan staining and controls (HPA tissue IHC; datasheet: 1:25).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
EPHA4 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 and membrane in cerebellum (HPA tissue IHC) | |
| Staining pattern | Cerebellar molecular layer: cytoplasm/membrane (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 | Adrenal gland+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Brain signal varies by cell type (HPA tissue IHC) | |
| Regulation | Expression regulation not specified (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope-side coverage is unknown (UniProt) |
Section 1
Recommended EPHA4 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is followed by published chromogenic IHC protocols for gastric adenocarcinoma, hippocampal tissue, and uveal melanoma (PMC3720259; PMC4149234; PMC9139903).
| Sample | Paraffin-embedded H. testis tissue; fixative not specified (datasheet A01860-1) |
| Fixation | Image fixative and duration unreported (datasheet A01860-1); 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-EPHA4, 1:25 (datasheet A01860-1) |
| 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 | EPHA4-positive staining in molecular layer cells - cytoplasm/membrane of cerebellum (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several different tissue types, highly abundant in brain. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval guidance); the gastric study used 98 °C for 20 min (PMC3720259).
Section 2
What Is the Expected EPHA4 Staining Pattern?
EPHA4 is a single-pass receptor with an extracellular region and cytoplasmic kinase region; UniProt places it at the cell membrane, neuronal projections, postsynaptic membrane, junctions and early endosomes (UniProt P54764 topology/localisation). In paraffin IHC, expect membrane-associated or cell-body staining in HPA-positive populations, especially brain neuropil, cerebellar molecular layer cells and placental syncytiotrophoblasts (HPA tissue IHC). HPA rates tissue staining reliability Enhanced, with medium consistency against RNA expression (HPA tissue IHC).
| Cerebral cortex neuropil stains strongly; cerebellar molecular layer cells show cytoplasmic/membrane staining. | These are reported High IHC patterns (HPA tissue IHC). Assess the labelled structures and surrounding cells separately; a strong field-wide deposit alone cannot establish the reported cell pattern (general IHC practice). |
| A population shows predominantly nuclear staining, with little convincing membrane or cytoplasmic signal. | Predominantly nuclear staining does not match the listed membrane, projection, junction or endosomal locations (UniProt P54764 localisation). Check morphology, the no-primary control and detection background before interpreting it as EPHA4 (general IHC practice). |
| Strong staining appears in an HPA-undetected cell population, such as adrenal glandular cells or bronchial respiratory epithelial cells. | HPA reports these specific populations as Not detected (HPA tissue IHC). Consider antibody cross-reactivity or endogenous detection activity; compare with an HPA-positive section and appropriate detection controls (general IHC practice). |
| Colour spreads across tissue, edges and spaces without a discernible cell or neuropil pattern. | This is difficult to score as EPHA4 because HPA reports staining in defined cells and neuropil (HPA tissue IHC). Examine no-primary and detection-only controls, washing and chromogen development for background (general IHC practice). |
| No stain is visible in cerebral cortex neuropil or placental syncytiotrophoblasts. | Both are High HPA IHC reference patterns (HPA tissue IHC). First confirm that the expected structures are present and the detection run worked; absence in one section does not establish EPHA4 absence (general IHC practice). |
💡Expected EPHA4 appearanceCall a positive result when staining resolves to High cortex neuropil, cerebellar molecular layer cell cytoplasm/membrane or placental syncytiotrophoblast cell bodies (HPA tissue IHC); isolated nuclear or structure-free colour is discordant with the reported pattern (UniProt P54764 localisation; HPA tissue IHC).
| Compartment and epitope position | EPHA4 spans the membrane at residues 548–569, with extracellular residues 20–547 and cytoplasmic residues 570–986 (UniProt P54764 topology). The supplied record gives no antibody epitope, so it cannot predict whether retrieval will favour a particular compartment or antibody (catalog antibody record). |
| Activation and intracellular signal | UniProt reports receptor clustering after activation and targeting to early endosomes (UniProt P54764 localisation). Thus a cellular component alongside membrane staining can fit the annotated biology; morphology and the HPA cell pattern remain necessary for interpretation (UniProt P54764 localisation; HPA tissue IHC). |
| Choice of reference tissue | Cortex neuropil, cerebellar molecular layer cells and placental syncytiotrophoblasts are High; colon enterocytes and kidney proximal tubule cell bodies are Medium (HPA tissue IHC). Use the same named population when comparing sections, since tissue-wide positivity would obscure these distinctions (general IHC practice). |
| Antibody evidence and limits | The supplied catalog antibody, CAB028368, is mouse monoclonal and IHC Enhanced (HPA antibodies). HPA describes overall tissue reliability as Enhanced but RNA agreement as medium consistency (HPA tissue IHC); judge a new pattern against controls rather than treating validation status as proof for every cell. |
| IF/ICC pattern? | HPA summarises EPHA4 as membrane-associated in its subcellular record, but supplies no main location or ICC-IF image cell lines (HPA subcellular). The IHC tissue patterns cannot establish an IF-positive cell line or IF protocol; consult the separate IF/ICC guide for that application (HPA tissue IHC; HPA subcellular). |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive brain or placenta is blank. | The slide may lack the named structure, or the staining run may have failed; HPA reports High signal only in specified populations (HPA tissue IHC; general IHC practice). | Confirm structure on the counterstain, check a concurrent positive-control section and review the run's retrieval, antibody and detection steps (general IHC practice). |
| Only faint signal appears in a nominally positive section. | The sampled population may differ from the HPA-positive one; staining levels also vary from High to Medium across listed populations (HPA tissue IHC). | Identify the precise cell type before changing conditions; compare it with a concurrent High reference and then review the run's validated settings (HPA tissue IHC; general IHC practice). |
| Nuclei dominate the stain. | A nuclear-dominant pattern is unsupported by the supplied EPHA4 localisation annotations (UniProt P54764 localisation); nonspecific or detection signal is possible (general IHC practice). | Inspect a no-primary control and compare membrane and cytoplasmic detail in an HPA-positive population before scoring the nuclei (general IHC practice; HPA tissue IHC). |
| Adrenal glandular cells or bronchial respiratory epithelium stain strongly. | HPA lists those populations as Not detected (HPA tissue IHC). Cross-reactivity or endogenous detection activity is a possibility, not a diagnosis from one slide (general IHC practice). | Run matched no-primary and detection controls, inspect localisation, and compare a positive reference population on the same staining run (general IHC practice; HPA tissue IHC). |
| Brown colour is diffuse or concentrated at tissue edges. | A deposit without the reported cell or neuropil pattern is hard to attribute to EPHA4 (HPA tissue IHC); excess detection background is possible (general IHC practice). | Review control slides, washing and chromogen development, then score only resolvable cellular or neuropil staining (general IHC practice; HPA tissue IHC). |
| The expected compartment changes between samples. | UniProt lists both membrane sites and early endosomes, with clustering after activation (UniProt P54764 localisation); the record gives no sample-specific mechanism for a shift. | Record membrane and cellular staining separately, verify the named HPA-positive cell population, and avoid assigning activation from localisation alone (UniProt P54764 localisation; HPA tissue IHC). |
Experiment evidence
IHC & IF images showing EPHA4 staining
A01860-1 · IHCImmunohistochemical analysis of paraffin-embedded H. testis section using EPHA4 Antibody (Center). A01860-1 was diluted at 1:25 dilution. A
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000116106-EPHA4/tissue/cerebral+cortex#imid_8602920
HPA · CerebellumHuman Protein Atlas IHC, Cerebellum. Source: https://www.proteinatlas.org/ENSG00000116106-EPHA4/tissue/cerebellum#imid_8602931
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000116106-EPHA4/tissue/colon#imid_8599334
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000116106-EPHA4/tissue/liver#imid_8599319
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for EPHA4 IHC & IF
🧪Run cerebellum first and look for staining in molecular layer cells at the cytoplasm or membrane (HPA: High in cerebellar molecular layer cells). Use adrenal gland as the negative tissue and check that its glandular cells remain unstained (HPA: Not detected in adrenal gland glandular cells); on the cerebellum slide, use only cells observed to lack signal as internal background references, since the supplied HPA row does not designate a negative cell type there.
Positive control tissue: Cerebellum (Molecular layer cells - cytoplasm/membrane, HPA High)
Negative control tissue: Adrenal gland (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for EPHA4; derive a cell-line control from the positive tissue's cell type (Molecular layer cells - cytoplasm/membrane) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary (secondary-only) control, an isotype control matched to the primary antibody’s host species, clonality and concentration, and EPHA4-knockout tissue where available as a biological negative (standard IHC practice). For chromogenic detection, block endogenous peroxidase and check the cerebellum slide for residual background before interpreting staining (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and retrieval dependence are unreported in the supplied evidence; optimize retrieval empirically (supplied target/application evidence). The selected A01860-1 caption shows paraffin-section DAB staining at 1:25, but its fixative is unreported (selected tissue-IHC caption). These data do not establish whether frozen sections or IF are easier; for IF in cerebellar neural tissue, assess autofluorescence with a no-primary control (standard IF practice).
HPA tissue IHC evidence for EPHA4
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 | Molecular layer cells - cytoplasm/membrane | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuropil | High | Protein (IHC) | HPA → |
| Placenta | Syncytiotrophoblasts - cell body | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Colon | Enterocytes | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Duodenum | Endocrine cells | Not detected | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced EPHA4 IHC Tips
Troubleshoot EPHA4 staining in paraffin sections by checking retrieval, antibody controls, expected localisation and cell type before interpreting chromogenic signal.
How should I retrieve EPHA4 when staining is weak in paraffin sections?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 minutes (page retrieval rule). Let sections cool consistently, then compare staining with a no-primary control and a positive control on the same run (standard IHC practice). If signal remains weak, test a second retrieval condition on adjacent sections while keeping antibody dilution and detection constant (standard IHC practice). Score whether the change improves cellular staining without increasing diffuse background, because EPHA4 can appear at the membrane and in cytoplasmic compartments (UniProt P54764 localisation; HPA tissue IHC). The selected antibody image establishes staining in a paraffin-embedded testis section but reports no retrieval method (A01860-1 caption).
Could fixation explain inconsistent EPHA4 staining across paraffin blocks?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative (A01860-1 caption). Record each block’s fixative and fixation duration, then compare sections processed with the same citrate pH 6.0, 95–98 °C, 20-minute retrieval and detection run (page retrieval rule; standard IHC practice). Include an internal tissue reference and a no-primary control so changes in antigen signal can be distinguished from changes in background (standard IHC practice). If staining differs between blocks, treat fixation as a possible processing variable rather than an established EPHA4-specific effect (standard IHC interpretation). Do not infer fixation sensitivity from EPHA4 topology or tissue expression (UniProt P54764 topology; HPA tissue IHC).
Is cytoplasmic EPHA4 staining credible for a membrane receptor?
Yes, cytoplasmic staining can be credible in context: tissue IHC reports cytoplasmic expression across several tissues, including high cytoplasm/membrane staining in cerebellar molecular layer cells (HPA tissue IHC). EPHA4 is also assigned to the cell membrane, neuronal projections, adherens junctions and early endosomes; activated receptor clusters and reaches early endosomes (UniProt P54764 localisation). Compare membrane-associated and cytoplasmic signal within the same cell population, then inspect adjacent sections for consistent anatomy (standard IHC practice). Do not require a sharp membrane rim in every positive cell, but investigate uniform haze extending across unrelated compartments with no-primary controls (HPA tissue IHC; standard IHC practice). Keep acquisition and counterstain settings comparable before assigning a compartment (standard IHC practice).
Can this antibody distinguish EPHA4 isoforms or extracellular from intracellular epitopes?
The supplied product caption identifies the antibody as “Center” but gives no epitope coordinates or isoform-specific validation (A01860-1 caption). EPHA4 has 2 listed isoforms, an extracellular region at residues 20–547, a transmembrane segment at 548–569 and a cytoplasmic region at 570–986 (UniProt P54764 topology; isoforms). Do not assign an isoform or membrane side from the staining pattern alone; obtain an epitope map or independent validation before making that claim (standard IHC interpretation). Extracellular glycosylation sites at 235, 340, 408 and 545 and cytoplasmic phosphorylation sites provide biologically relevant context, but their effects on this antibody’s staining are unknown (UniProt P54764 modifications; A01860-1 caption).
How can I check an EPHA4 IHC pattern with multiplex immunofluorescence?
Use IF on a matched section as an orthogonal localisation check, while treating it as a separate assay because the supplied antibody caption documents chromogenic paraffin-section IHC only (A01860-1 caption; standard IHC/IF practice). Pair EPHA4 with an independently validated marker for the cell population under study; compare the overlap with the expected neuropil or cellular compartments rather than fluorescence intensity alone (HPA tissue IHC; standard IF practice). Select fluorophores after measuring tissue autofluorescence, favouring channels with lower background in that specimen (standard IF practice). If the mapped epitope is cytoplasmic, optimise permeabilisation; if extracellular, first assess staining without permeabilisation, since the supplied epitope side is unspecified (UniProt P54764 topology; A01860-1 caption; standard IF practice).
How do I separate weak EPHA4 staining from chromogenic background?
Run no-primary and secondary-only controls, and inspect the same tissue region for diffuse precipitate or endogenous staining before scoring EPHA4 (standard IHC practice). The selected image used antibody A01860-1 at 1:25, a biotinylated secondary and DAB, but supplies no generalisable background threshold (A01860-1 caption). Apply a peroxidase block and assess endogenous biotin where relevant to the detection system; these are general chromogenic controls, not EPHA4-specific findings (standard IHC practice). Titrate primary antibody and detection while holding retrieval at citrate pH 6.0, 95–98 °C for 20 minutes (page retrieval rule; standard IHC practice). Compare suspected signal with the tissue’s cell pattern and control sections (HPA tissue IHC; standard IHC practice).
What is a defensible way to quantify EPHA4 IHC across samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the anatomical region and eligible cell population before scoring, then use either percent-positive cells, an intensity-weighted H-score, or positive area density per mm² (standard IHC quantification). Normalise positive counts to eligible cells and area measurements to viable tissue area, excluding folds, edges and necrotic regions by a fixed rule (standard IHC quantification). Record membrane-associated and cytoplasmic staining separately where resolution permits, since both are plausible for EPHA4 (UniProt P54764 localisation; HPA tissue IHC). Keep retrieval, antibody dilution, detection exposure and counterstain consistent across compared sections; the selected image used 1:25 but does not establish an optimal analytical dilution (A01860-1 caption; standard IHC practice).
When should I question an apparent EPHA4-positive tissue compartment?
Check whether signal follows plausible cellular or neuropil structures: high staining is reported in cerebral cortex neuropil and cerebellar molecular layer cells, while EPHA4 is assigned to membranes, projections, junctions and early endosomes (HPA tissue IHC; UniProt P54764 localisation). Staining confined to section edges, folds, necrosis or a no-primary control is more consistent with artefact than specific EPHA4 detection (standard IHC interpretation). Likewise, unexpected staining in a cell population reported as undetected by HPA should prompt control review, not automatic rejection, because its tissue-IHC reliability is “Enhanced” with medium RNA–protein consistency (HPA tissue IHC). Recheck retrieval, secondary-only controls and endogenous peroxidase before interpreting DAB deposits as receptor localisation (page retrieval rule; standard IHC practice).
Boster reagents
Best EPHA4 / Ephrin type-A receptor 4 IHC Antibodies
A01860-1 has real IHC data from a human paraffin-embedded testis section (image caption); the catalog lists human and mouse reactivity (catalog: reactivity).
Real IHC data
Anti-EPHA4 Antibody (Center)
Cat # A01860-1
A01860-1 will render with an IHC image of a human paraffin-embedded testis section stained at 1:25 with DAB detection (image caption). Its listed applications are IHC-P and WB, and its listed reactivity is human and mouse (catalog: applications and reactivity).
Which to pick: Choose A01860-1 for paraffin-section IHC: its own image shows staining of a human testis section (image caption). The caption does not report the fixative (image caption). For mouse tissue, A01860-1 has listed mouse reactivity but no mouse IHC image in the payload (catalog: reactivity; image caption); no IF/ICC application or IF image is listed for this SKU (catalog: applications and images).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P54764 (EPHA4_HUMAN, Ephrin type-A receptor 4).
- Human Protein Atlas. EPHA4 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. EPHA4 subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. EPHA4 antibody validation summary (1 antibodies).
- EphA4 is a prognostic factor in gastric cancer. BMC clinical pathology 2013 — PMC3720259.
- Altered distribution of the EphA4 kinase in hippocampal brain tissue of patients with Alzheimer's disease correlates with pathology. Acta neuropathologica communications 2014 — PMC4149234.
- Generation of an EphA4 conditional allele in mice. Genesis (New York, N.Y. : 2000) 2010 — PMC2819605.
- EPHA2, EPHA4, and EPHA6 Expression in Uveal Melanomas: Searching for the Culprits of Neoplasia. Diagnostics (Basel, Switzerland) 2022 — PMC9139903.
- PubMed PMID:7898931 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15815621 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for EPHA4 (Ephrin type-A receptor 4). Expected localisation Cytoplasm and membrane in cerebellum (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A01860-1 at 1:25 (the product's own IHC validation image is shown). Controls (HPA): positive Cerebellum (Molecular layer cells - cytoplasm/membrane); negative Adrenal gland. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded H. testis tissue; fixative not specified (datasheet A01860-1); 2. Fixation: Image fixative and duration unreported (datasheet A01860-1); 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-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-EPHA4, 1:25 (datasheet A01860-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Brain signal varies by cell type (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope-side coverage is unknown (UniProt); Regulation: Expression regulation not specified (UniProt). Evidence: UniProt P54764, HPA tissue IHC · Enhanced, HPA ICC-IF · Membrane, Datasheet A01860-1, PMC3720259, PMC4149234. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/EPHA4/immunohistochemistry-ihc-guide.