EPHA3 / Ephrin type-A receptor 3 · IHC design guide

Design Immunohistochemistry for EPHA3

Plan paraffin EPHA3 IHC with the catalog antibody at 2–5 μg/ml (datasheet A02872-1). Compare kidney tubule cells with skeletal muscle myocytes, and score the cytoplasmic tissue pattern alongside the expected membrane location (HPA tissue IHC; UniProt).

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

Immunohistochemistry protocol sheet for EPHA3 (IHC for EPHA3): expected localisation Cytoplasmic tissue staining (HPA tissue IHC); membrane expected (UniProt), antibody A02872-1, validated IHC image, and IHC protocol steps — Printable **EPHA3** IHC protocol sheet — expected localisation Cytoplasmic tissue staining (HPA tissue IHC); membrane expected (UniProt), antibody A02872-1, controls and protocol steps. Open the full EPHA3 IHC guide →

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EPHA3 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 tissue staining (HPA tissue IHC); membrane expected (UniProt)
	Staining pattern	Cytoplasmic staining in cells of most tissues (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A02872-1)
	Positive control ⓘ	Adipose tissue+4 more · see all
	Negative control ⓘ	Appendix+4 more · see all

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific)
	Caveat	Antibody staining has low consistency with RNA expression (HPA tissue IHC)
	Regulation	Highest expression in placenta (UniProt)
	Isoform / epitope	2 isoforms; check which side of the membrane contains the epitope (UniProt)

Section 1

Recommended EPHA3 IHC & IF Protocols

The catalog antibody’s IHC-P protocol (datasheet: A02872-1) is accompanied by published EPHA3 staining methods for gastric cancer, colorectal cancer, FFPE cell blocks, and mouse lung (PMC5408411; PMC5294649; PMC12272597; PMC4381338).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human placenta tissue; fixative not specified (datasheet A02872-1)
Fixation	Image fixative and duration unreported (datasheet A02872-1); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat retrieval: EDTA pH 8.0 (datasheet A02872-1); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A02872-1)
Primary antibody	Rabbit anti-EPHA3, 2-5 μg/ml (datasheet A02872-1)
Primary incubation	Overnight at 4 °C (datasheet A02872-1)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet A02872-1)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	EPHA3-positive staining in adipocytes of adipose tissue (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA at pH 8.0 for the catalog antibody (datasheet: A02872-1); published citrate and trypsin retrieval conditions belong to the antibodies used in those studies (PMC5408411; PMC4381338).

Section 2

What Is the Expected EPHA3 Staining Pattern?

EPHA3 is a membrane receptor with an extracellular region and a cytoplasmic tail (UniProt P29320 topology). In tissue IHC, HPA reports predominantly cytoplasmic staining across most tissues, including medium staining in kidney tubule cells, cardiomyocytes and several epithelial or neuronal cell populations (HPA tissue IHC). HPA rates its tissue staining Approved, while noting low consistency with RNA expression (HPA tissue IHC reliability).

What am I looking at on my slide?
Cytoplasmic staining in kidney tubule cells or cardiomyocytes, with some cell-edge accentuation.	Medium staining in these cells matches HPA tissue observations (HPA tissue IHC). Cell-edge signal is plausible for a transmembrane receptor (UniProt P29320 topology), but HPA's tissue profile is chiefly cytoplasmic (HPA tissue IHC).
Strong, isolated nuclear staining dominates the chromogenic section.	This does not match HPA's predominant tissue IHC profile and warrants scrutiny (HPA tissue IHC). ICC-IF reports some nucleoplasmic localisation, so nuclear signal alone cannot establish either an artefact or a valid tissue pattern (HPA subcellular ICC-IF).
Staining appears in bone marrow hematopoietic cells or pancreatic exocrine glandular cells.	HPA reports EPHA3 as not detected in these cells (HPA tissue IHC). Check for cross-reactivity or, with enzyme-based detection, endogenous enzyme activity before interpreting the signal as EPHA3; the HPA result is a reference pattern, not proof of absence.
Diffuse colour covers cells and surrounding tissue without a clear cellular boundary.	This is difficult to score against HPA's cellular staining profile (HPA tissue IHC). Review background controls, blocking and washing; diffuse colour alone does not identify EPHA3-positive cells.
No staining appears in kidney tubule cells in an otherwise evaluable section.	HPA reports medium staining in these cells (HPA tissue IHC). Review the IHC-validated antibody, retrieval and detection controls before calling the sample negative; HPA's low RNA–staining consistency also limits conclusions from a single section (HPA tissue IHC reliability).

💡Expected EPHA3 appearanceA convincing positive is discernible, chiefly cytoplasmic staining of an HPA medium-positive cell population, such as kidney tubule cells (HPA tissue IHC); diffuse colour or strong staining of HPA not-detected cells calls for control review (HPA tissue IHC).

How each factor affects the staining
Where should the IHC signal sit?	EPHA3 spans the membrane, with residues 21–541 extracellular and 566–983 cytoplasmic (UniProt P29320 topology). HPA nevertheless describes most tissue IHC staining as cytoplasmic (HPA tissue IHC); interpret membrane accentuation alongside that observed pattern.
How strong is the tissue reference?	HPA rates the tissue IHC profile Approved but reports low consistency between antibody staining and RNA expression (HPA tissue IHC reliability). Its cell-level observations guide comparison; they do not independently confirm every stained cell.
Can isoforms or processing change interpretation?	UniProt lists two isoforms and a signal peptide at residues 1–20 (UniProt P29320 isoforms and processing). The supplied evidence gives no antibody epitope, so it cannot establish which isoforms this IHC assay detects.
What does the ICC-IF pattern suggest?	HPA reports nuclear membrane, plasma membrane and cytosol as main ICC-IF locations, with nucleoplasm and actin filaments additionally observed (HPA subcellular ICC-IF). These cell-image findings provide context, not a paraffin IHC pattern or an IF protocol.
Does the record specify EPHA3 retrieval sensitivity?	No target-specific fixation or retrieval effect is supplied. Optimise antigen retrieval using routine IHC controls; neither HPA tissue staining nor UniProt topology establishes that a particular retrieval condition changes EPHA3 detection.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
The expected kidney tubule signal is absent.	A failed staining run or unsuitable assay conditions are possible; HPA reports medium staining in kidney tubule cells (HPA tissue IHC).	Check a positive control, antibody incubation, retrieval and detection steps before scoring the section negative.
Chromogen appears in HPA not-detected cells.	Cross-reactivity or endogenous detection activity may contribute; HPA reports no signal in bone marrow hematopoietic cells and pancreatic exocrine glandular cells (HPA tissue IHC).	Compare an antibody-omission control and, if using peroxidase detection, an appropriate endogenous-peroxidase control.
The section has broad, hazy background.	Non-specific binding, inadequate washing or overdeveloped chromogen may obscure cellular staining; HPA describes a cellular tissue profile (HPA tissue IHC).	Review blocking, washes and development time against a control section; score only resolvable cellular signal.
Only nuclei show strong IHC signal.	This differs from the chiefly cytoplasmic tissue IHC profile (HPA tissue IHC), although nucleoplasmic signal is reported in ICC-IF (HPA subcellular ICC-IF).	Check morphology and background controls, then avoid assigning the nuclear-only pattern to EPHA3 from IHC appearance alone.
Staining varies among tissue regions or cell types.	EPHA3 staining is cell-dependent in the HPA tissue panel, and HPA notes low RNA–staining consistency (HPA tissue IHC).	Score named cell populations separately, using matched morphology and controls rather than a whole-section average.
Membrane staining is weak despite clear cytoplasmic colour.	HPA describes cytoplasmic expression in most tissues (HPA tissue IHC), while UniProt places EPHA3 at the cell membrane (UniProt P29320 subcellular location).	Record the observed compartment and cell type; do not reject an otherwise control-supported tissue pattern solely because membrane outlining is faint.

Source: UniProt P29320 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing EPHA3 staining

IHC analysis of Eph Receptor A3/EPHA3 using anti-Eph Receptor A3/EPHA3 antibody (A02872-1). Eph Receptor A3/EPHA3 was detected in a paraffin-embedded section of human placenta tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-Eph Receptor A3/EPHA3 Antibody (A02872-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

A02872-1 · IHCIHC analysis of Eph Receptor A3/EPHA3 using anti-Eph Receptor A3/EPHA3 antibody (A02872-1). Eph Receptor A3/EPHA3 was detected in a paraffin

IF analysis of Eph Receptor A3/EPHA3 using anti-Eph Receptor A3/EPHA3 antibody (A02872-1). Eph Receptor A3/EPHA3 was detected in an immunocytochemical section of PC-3 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-Eph Receptor A3/EPHA3 Antibody (A02872-1) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

A02872-1 · IFIF analysis of Eph Receptor A3/EPHA3 using anti-Eph Receptor A3/EPHA3 antibody (A02872-1). Eph Receptor A3/EPHA3 was detected in an immunocy

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000044524-EPHA3/tissue/cerebral+cortex#imid_2723020

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000044524-EPHA3/tissue/colon#imid_2722938

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000044524-EPHA3/tissue/liver#imid_2722939

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000044524-EPHA3/tissue/kidney#imid_2722948

HPA · ICC-IF AF22Human Protein Atlas immunofluorescence, AF22, antibody HPA069390; green = EPHA3, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for EPHA3 IHC & IF

🧪Run bronchus first: respiratory epithelial cells should stain at the HPA Medium level (HPA: bronchus, respiratory epithelial cells). Use appendix glandular cells as the negative tissue (HPA: appendix, glandular cells, Not detected); on the bronchus slide, neighboring cells without specific staining should remain at background, without assuming a particular cell type is negative.

Positive control tissue: Adipose tissue (Adipocytes, HPA Medium)

Negative control tissue: Appendix (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show EPHA3 in AF22, U-251MG, U2OS, with annotated localisation: Nuclear membrane (approved), Plasma membrane (supported), Cytosol (approved) (HPA subcellular).

Technical controls: Include a no-primary (secondary-only) control, a concentration-matched rabbit IgG control matched to the primary antibody’s class and clonality where known (IHC caption: rabbit primary antibody), and an EPHA3 knockout control or cognate peptide block if available. For HRP/DAB staining of bronchus, quench endogenous peroxidase and assess background alongside epithelial signal (IHC caption: HRP/DAB detection; HPA: bronchus, respiratory epithelial cells).

⚠️Feasibility: The selected A02872-1 paraffin-section caption uses heat retrieval in EDTA at pH 8.0, but reports no fixative; no target-specific fixation window or fixation effect is reported (IHC caption: EDTA retrieval; fixative unreported). This supports heat retrieval as a starting condition, without establishing whether it is required (IHC caption: heat retrieval). The supplied evidence does not establish that frozen sections or IF are easier; ICC-IF images are available for AF22, U-251MG and U2OS (HPA: subcellular ICC-IF images).

HPA tissue IHC evidence for EPHA3

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low 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
Adipose tissue	Adipocytes	Medium	Protein (IHC)	HPA →
Bronchus	Respiratory epithelial cells	Medium	Protein (IHC)	HPA →
Caudate	Neuronal cells	Medium	Protein (IHC)	HPA →
Cerebral cortex	Neuronal cells	Medium	Protein (IHC)	HPA →
Duodenum	Glandular cells	Medium	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Appendix	Glandular cells	Not detected	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	Not detected	Protein (IHC)	HPA →
Pancreas	Exocrine glandular cells	Not detected	Protein (IHC)	HPA →
Skeletal muscle	Myocytes	Not detected	Protein (IHC)	HPA →
Skin	Fibroblasts	Not detected	Protein (IHC)	HPA →

Section 3

Advanced EPHA3 IHC Tips

Troubleshoot EPHA3 staining in paraffin sections by checking retrieval, compartment, controls and scoring before interpreting a chromogenic signal.

How should I retrieve EPHA3 in paraffin sections when staining is weak?

Start with heat-mediated retrieval in EDTA at pH 8.0 for the IHC-validated antibody (datasheet A02872-1). The demonstrated paraffin-section workflow used 2 μg/ml primary antibody overnight at 4°C, so keep those conditions consistent while assessing retrieval (datasheet A02872-1). If staining remains weak, compare retrieval duration on matched sections while keeping the EDTA buffer and detection steps fixed (standard IHC practice). Check a human placenta section alongside the test tissue because that is the tissue shown in the selected antibody’s IHC caption (datasheet A02872-1). Avoid treating a stronger signal alone as proof of specificity; inspect its cell distribution and compartment (standard IHC practice).

Could fixation explain variable EPHA3 staining across paraffin blocks?

The selected paraffin-section caption does not state the fixative, so target-specific fixation sensitivity is unknown (datasheet A02872-1). Record each block’s fixative and processing history, and compare sections with similar handling before attributing a signal difference to EPHA3 abundance (standard IHC practice). Run the same EDTA pH 8.0 retrieval and 2 μg/ml primary concentration across that comparison (datasheet A02872-1). If archival blocks differ in staining, examine tissue preservation and internal morphology before changing antibody conditions (standard IHC practice). Neither the reported tissue pattern nor EPHA3 topology establishes which fixation conditions preserve this antibody’s epitope (HPA: tissue IHC profile; UniProt P29320 topology).

Should EPHA3 staining be membranous, cytoplasmic or nuclear?

Interpret membrane staining in light of EPHA3’s extracellular residues 21–541, transmembrane segment 542–565 and cytoplasmic residues 566–983 (UniProt P29320 topology). Cytoplasmic IHC staining is also reported across most tissues, so a cytoplasmic pattern alone does not exclude EPHA3 (HPA: tissue IHC profile). Subcellular imaging reports plasma membrane, nuclear membrane and cytosol as main locations, with additional nucleoplasm and actin-filament localisation (HPA: subcellular). Compare compartments within intact cells and against a no-primary control before assigning diffuse DAB deposits to the receptor (standard IHC practice). Document the scored compartment explicitly, because membrane and cytoplasmic scores answer different biological questions (standard IHC practice).

How does an unknown epitope affect interpretation of EPHA3 staining?

EPHA3 has 2 recorded isoforms, but the selected caption does not identify this antibody’s epitope or establish isoform specificity (UniProt P29320 isoforms; datasheet A02872-1). Its extracellular region spans residues 21–541, while the cytoplasmic region spans 566–983; these regions require different access in intact cells (UniProt P29320 topology; standard IF practice). Five reported glycosylation sites lie in the extracellular region, and reported phosphotyrosines lie on the cytoplasmic side (UniProt P29320 modifications). Consult a documented immunogen or epitope before claiming that staining distinguishes isoforms, glycosylation states or phosphorylation states (standard antibody-validation practice). Until then, describe the result as EPHA3 immunoreactivity in the observed compartment (standard IHC practice).

How can I check EPHA3 localisation by multiplex IF?

Use a validated marker for the cell population being evaluated, and inspect EPHA3 and marker channels separately before judging overlap (standard IF practice). EPHA3 has extracellular residues 21–541 and cytoplasmic residues 566–983, but the selected IHC caption does not identify the antibody epitope (UniProt P29320 topology; datasheet A02872-1). For an extracellular epitope, assess membrane access without unnecessary permeabilisation; for an intracellular epitope, optimise controlled permeabilisation after fixation (standard IF practice). Choose a far-red fluorophore when tissue autofluorescence obscures shorter-wavelength channels, and include single-channel and no-primary controls (standard IF practice). Treat IF localisation as a separate validation exercise because the supplied antibody evidence describes paraffin-section chromogenic IHC (datasheet A02872-1).

What controls help distinguish EPHA3 staining from DAB background?

The demonstrated workflow blocked sections with 10% goat serum, then used a peroxidase-conjugated secondary and DAB detection (datasheet A02872-1). Include a no-primary section to reveal secondary-reagent or detection background, and apply an endogenous peroxidase block as a general chromogenic IHC step (standard IHC practice). Examine whether deposits follow intact cell boundaries or collect at section folds, damaged edges and necrotic areas (standard IHC practice). Keep DAB development and counterstaining consistent across comparison sections so intensity differences remain interpretable (standard IHC practice). If background persists, assess blocking, washes and secondary specificity while retaining a matched positive section (standard IHC practice).

How should I score EPHA3 across sections with uneven staining? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and compartment before scoring, because EPHA3 has reported membrane and cytosolic localisation (HPA: subcellular). For chromogenic sections, record the percentage of positive target cells and staining intensity, or calculate an H-score from intensity categories and their cell percentages (standard IHC scoring practice). Normalise positive-cell counts to the number of eligible cells, or stained-cell density to assessed tissue area in mm² (standard IHC scoring practice). Exclude folds, necrosis and poorly preserved regions using the same rules for every section (standard IHC practice). Report membrane and cytoplasmic results separately, with matched exposure to retrieval and DAB development conditions (standard IHC practice).

When is an EPHA3-positive IHC pattern convincing rather than artefactual?

Look for reproducible staining in intact cells with a defined compartment; EPHA3 is reported at the plasma membrane and in the cytosol (HPA: subcellular). Compare the sampled cell type with tissue context: HPA reports medium staining in kidney tubule cells but no detection in skeletal-muscle myocytes (HPA: tissue IHC). Signal limited to cut edges, folds or necrosis warrants caution, and no-primary staining can expose endogenous enzyme or detection background (standard IHC practice). A placenta section can check the selected antibody workflow because the product caption documents paraffin-section EPHA3 detection there (datasheet A02872-1). Interpret unexpected patterns cautiously because the HPA tissue IHC profile has low consistency with RNA expression (HPA: reliability description).

Boster reagents

Best EPHA3 / Ephrin type-A receptor 3 IHC Antibodies

A02872-1 has human paraffin-section IHC data from placenta and IF/ICC data from PC-3 cells (catalog image captions).

Real IHC data

Anti-Eph receptor A3/EPHA3 Antibody ®

Cat # A02872-1

View product →

A02872-1 was tested for IHC on a human placenta paraffin section (catalog IHC image caption). The same SKU was tested for IF/ICC in PC-3 cells (catalog IF image caption).

Which to pick: Choose A02872-1 for human tissue IHC when using paraffin sections; its IHC image documents placenta staining, but does not report the fixative (catalog IHC image caption). For IF/ICC, A02872-1 has a PC-3 cell image and a listed working concentration of 5 μg/ml (catalog IF image caption; datasheet). No cross-species choice is supported: A02872-1 lists human reactivity only, and its clonality is unreported (catalog reactivity and clone fields).

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

References

UniProt Consortium. UniProt entry P29320 (EPHA3_HUMAN, Ephrin type-A receptor 3).
Human Protein Atlas. EPHA3 tissue IHC expression (reliability: Approved).
Human Protein Atlas. EPHA3 subcellular location (ICC-IF): Mainly localized to the nuclear membrane, plasma membrane and cytosol. In addition localized to the nucleoplasm and actin filaments..
Human Protein Atlas. EPHA3 antibody validation summary (2 antibodies).
High expression of EphA3 (erythropoietin-producing hepatocellular A3) in gastric cancer is associated with metastasis and poor survival. BMC clinical pathology 2017 — PMC5408411.
Investigation of the role of tyrosine kinase receptor EPHA3 in colorectal cancer. Scientific reports 2017 — PMC5294649.
Development of a specific anti-human EphA3 monoclonal antibody, Ea(3)Mab-20, for flow cytometry. Biochemistry and biophysics reports 2025 — PMC12272597.
The putative tumor suppressor gene EphA3 fails to demonstrate a crucial role in murine lung tumorigenesis or morphogenesis. Disease models & mechanisms 2015 — PMC4381338.
PubMed PMID:1311845 — UniProt-cited evidence.
PubMed PMID:10987298 — UniProt-cited evidence.
PubMed PMID:1737782 — UniProt-cited evidence.