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EPHA1 / Ephrin type-A receptor 1 · IHC design guide
Design Immunohistochemistry for EPHA1
Plan EPHA1 paraffin-section IHC around the cytoplasmic and membranous staining reported in most tissues (HPA tissue IHC). Use glandular cells with medium staining as a reference and account for epitope location when interpreting results (HPA tissue IHC; UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
EPHA1 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 | Membranous and cytoplasmic in most tissues (HPA tissue IHC) | |
| Staining pattern | Most tissues show cytoplasmic and membranous staining (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Smooth muscle |
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 PA1573) | |
| Caveat | Phospho-Tyr605 staining depends on site phosphorylation (UniProt; catalog A03024Y605-1) | |
| Regulation | Overexpressed in several carcinomas (UniProt) | |
| Isoform / epitope | 3 isoforms; map epitopes to extracellular or cytoplasmic regions (UniProt) |
Section 1
Recommended EPHA1 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with three published EPHA1 IHC methods (PMC7133419; PMC4234387; PMC8700455).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet PA1573); 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-EPHA1, 0.5-1μg/ml (datasheet PA1573) |
| 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 | EPHA1-positive staining in glandular cells of adrenal gland (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic and membranous expression in most tissues, including parathyroid gland. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page antigen retrieval rule).
Section 2
What Is the Expected EPHA1 Staining Pattern?
EPHA1 is a single-pass cell-membrane receptor with an extracellular region at residues 26–547 and a cytoplasmic region at 569–976 (UniProt P21709 topology). In paraffin-section IHC, expect membranous staining with possible cytoplasmic staining in epithelial or glandular cells; HPA reports this pattern across most tissues (HPA tissue IHC). Treat the pattern as provisional: HPA rates the tissue IHC reliability Uncertain (HPA tissue IHC).
| Membrane-associated staining in glandular or respiratory epithelial cells, with some cytoplasmic signal. | This fits the reported cytoplasmic and membranous tissue pattern (HPA tissue IHC) and EPHA1 cell-membrane location (UniProt P21709). Compare cells within the same section: HPA reports Medium staining in adrenal, appendix, breast, and cervix glandular cells and bronchial respiratory epithelial cells (HPA tissue IHC). |
| Strong nuclear-only staining, with little signal at cell borders or in cytoplasm. | Nuclear-only staining does not fit the reported membrane location (UniProt P21709; HPA subcellular summary) or the tissue IHC pattern (HPA tissue IHC). Check morphology and staining controls before assigning this signal to EPHA1; the antibody's tissue IHC reliability is Uncertain (HPA tissue IHC). |
| Staining is strongest in a cell population expected to be unstained, while the expected cells are faint. | For example, HPA reports smooth muscle cells as Not detected and bronchial respiratory epithelial cells as Medium (HPA tissue IHC). A reversed pattern raises concern for cross-reactivity or endogenous chromogenic detection activity; verify the cell identities and examine a no-primary control. |
| Uniform color covers tissue and spaces between cells, obscuring cell borders. | Diffuse background cannot establish EPHA1 localization. In chromogenic IHC, inspect a no-primary control for detection-related signal, then review blocking, antibody concentration, washes, and imaging exposure as general workflow checks. HPA's Uncertain reliability makes a clear cellular pattern especially useful (HPA tissue IHC). |
| No staining appears in a comparison section selected for reported EPHA1 staining. | HPA reports Medium staining in several glandular and epithelial cell populations, but its tissue IHC reliability is Uncertain (HPA tissue IHC). Confirm that the sampled section contains the specified cells, then check the primary antibody and detection run. One negative section alone cannot distinguish absent antigen from assay failure. |
💡Expected EPHA1 appearanceCall a result consistent with EPHA1 when membranous, possibly cytoplasmic, staining is discernible in the reported glandular or epithelial cells at roughly the HPA-reported Medium level; nuclear-only or uniform background staining is suspect, and HPA rates the IHC pattern Uncertain (UniProt P21709; HPA tissue IHC).
| Compartment and unknown antibody epitope | EPHA1 spans the membrane, with extracellular residues 26–547 and cytoplasmic residues 569–976 (UniProt P21709 topology). The supplied antibody record gives no epitope, so topology cannot predict which domain CAB026144 recognizes or specify an EPHA1-specific antigen-retrieval condition. |
| Tissue and cell selection | HPA reports Medium staining in listed glandular, respiratory epithelial, and selected neural cells; smooth muscle cells are Not detected (HPA tissue IHC). Score the named cell population rather than treating every cell in a tissue as equally informative. These are reported patterns, not guaranteed controls (HPA reliability: Uncertain). |
| Antibody validation | CAB026144 is a mouse monoclonal antibody with IHC status Uncertain and no ICC status supplied (HPA antibodies). The tissue profile also has Uncertain reliability (HPA tissue IHC). Interpret weak or unexpected staining against tissue morphology and controls; no independently confirmed IHC pattern is supplied here. |
| IF/ICC expectation? | HPA summarizes EPHA1 as Membrane, but supplies no main ICC-IF location or cell lines with ICC-IF images (HPA subcellular). Membrane localization is a reasonable expectation (UniProt P21709; HPA subcellular), but this record cannot validate an IF/ICC staining pattern or supply an IF/ICC protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| Reported Medium-staining cells are blank in paraffin-section IHC. | The section may lack the named cells, or the antibody and chromogenic detection run may have failed; the reported level is provisional (HPA tissue IHC: Medium; reliability Uncertain). | Confirm cell identity and tissue preservation on the counterstained section. Run a documented positive assay control, then review the primary-antibody, retrieval, and detection steps as general IHC workflow checks; no EPHA1-specific retrieval condition is supplied. |
| Only nuclei stain. | Nuclear-only staining conflicts with EPHA1's reported membrane location and tissue IHC pattern (UniProt P21709; HPA tissue IHC). Its source remains unresolved from appearance alone. | Check the no-primary control and review nuclear counterstain separately. Reassess whether membrane or cytoplasmic staining is present in the HPA-reported cells before interpreting the nuclear signal as EPHA1 (HPA tissue IHC). |
| Smooth muscle cells stain more strongly than adjacent reported positive cells. | HPA lists smooth muscle cells as Not detected (HPA tissue IHC). Cross-reactivity or endogenous chromogenic activity is possible; staining alone does not identify which. | Verify cell boundaries and identities, inspect a no-primary control, and compare with a section containing HPA-reported Medium cells (HPA tissue IHC). If the unexpected pattern persists, treat the antibody result as unresolved. |
| Diffuse brown signal obscures cell borders. | Background may arise during general chromogenic IHC detection or from excessive nonspecific staining; it does not demonstrate EPHA1's membrane location (UniProt P21709). | Inspect no-primary and reagent controls. Review blocking, primary-antibody concentration, wash stringency, and chromogen development as general IHC steps; retain only cell-associated staining for interpretation. |
| Cytoplasmic staining appears without a sharp membrane rim. | HPA reports cytoplasmic as well as membranous tissue expression (HPA tissue IHC), so cytoplasmic signal alone is not proof of an artefact. The supplied record does not establish the antibody epitope or a processing explanation. | Check whether staining follows the HPA-reported cell distribution and whether background controls are clean (HPA tissue IHC). Record the compartment honestly; avoid calling a weak or diffuse cytoplasmic result confirmed membrane staining. |
| An IF/ICC image shows an unexpected distribution. | The supplied HPA ICC-IF record has a Membrane summary but no imaged cell lines or main location, and CAB026144 has no ICC status supplied (HPA subcellular; HPA antibodies). | Treat the IF/ICC result as unvalidated by this IHC evidence. Assess it with controls and the dedicated IF/ICC guide; do not transfer an IHC-P interpretation or protocol to IF/ICC. |
Experiment evidence
IHC & IF images showing EPHA1 staining
PA1573 · IHCAnti-Eph receptor A1 antibody, PA1573, IHC(P) IHC(P): Rat Brain Tissue
PA1573 · IHCAnti-Eph receptor A1 antibody, PA1573, IHC(P) IHC(P): Human Colon Cancer Tissue
A03024Y605-1 · IHCImmunohistochemical analysis of paraffin-embedded human tonsil. 1, Antibody was diluted at 1:200 (4° overnight). 2, Tris-EDTA, pH9.0 was use
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000146904-EPHA1/tissue/cerebral+cortex#imid_6997368
HPA · Parathyroid glandHuman Protein Atlas IHC, Parathyroid gland. Source: https://www.proteinatlas.org/ENSG00000146904-EPHA1/tissue/parathyroid+gland#imid_6997267
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000146904-EPHA1/tissue/colon#imid_6997310
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000146904-EPHA1/tissue/liver#imid_6997311
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for EPHA1 IHC & IF
🧪Run adrenal gland first: glandular cells should stain at the HPA Medium level (HPA: adrenal gland, glandular cells, Medium); run smooth muscle as the negative tissue (HPA: smooth muscle cells, Not detected). On the adrenal slide, use cells outside the glandular compartment as internal background comparators, while confirming their EPHA1 status independently (HPA: adrenal gland, glandular cells, Medium).
Positive control tissue: Adrenal gland (Glandular cells, HPA Medium)
Negative control tissue: Smooth muscle (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for EPHA1; derive a cell-line control from the positive tissue's cell type (Glandular cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control; a host-, immunoglobulin-class-, and clonality-matched irrelevant antibody control; and EPHA1-knockout tissue as a biological negative when available (standard IHC practice). Check endogenous peroxidase in adrenal sections and block endogenous biotin if using avidin–biotin detection (standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected paraffin-section caption does not state a fixative (PA1573 caption: IHC(P), Rat Brain Tissue; fixative unreported). Antigen retrieval dependence and adrenal-specific artefacts are unreported; compare retrieval conditions empirically and inspect background in the adrenal section (HPA: adrenal gland, glandular cells, Medium). Whether frozen sections or IF/ICC are easier is unreported, and the supplied HPA subcellular record lists no cell lines with ICC-IF images (HPA subcellular: Membrane; no ICC-IF image cell lines).
HPA tissue IHC evidence for EPHA1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — External characterization data supports antibody staining.). 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Caudate | Glial cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced EPHA1 IHC Tips
Troubleshoot EPHA1 staining in paraffin section chromogenic IHC using its membrane topology (UniProt P21709) and tissue staining evidence rated Uncertain (HPA tissue IHC).
What should I change when EPHA1 staining is weak after retrieval?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval setting). Check that sections remain covered by buffer, cool them consistently, and compare a retrieval time series against a section processed with the page setting (standard IHC practice). Assess membranous staining separately from diffuse cytoplasmic colour because EPHA1 is a cell membrane receptor, while tissue IHC reports both patterns (UniProt P21709; HPA tissue IHC). If signal remains weak, trial an alternative retrieval buffer or pH on matched sections, changing one condition at a time (standard IHC practice). Keep exposure and detection conditions identical across that comparison (standard IHC practice).
Could fixation explain weak or patchy EPHA1 staining in paraffin sections?
EPHA1-specific sensitivity to fixative type or fixation duration is unknown from the supplied evidence (supplied IHC evidence). The PA1573 image identifies rat brain tissue and IHC(P), but its caption does not state the fixative (PA1573 tissue-IHC caption). Compare sections prepared with documented fixation and processing histories, holding retrieval at citrate pH 6.0, 95–98 °C for 20 min (page retrieval setting; standard IHC practice). Inspect tissue preservation alongside staining before attributing patchiness to antigen loss (standard IHC practice). Record fixation duration and section thickness for each comparison, and repeat staining with matched detection settings (standard IHC practice).
How should I assess membrane versus cytoplasmic EPHA1 colour?
Score membrane-associated colour first because EPHA1 has an extracellular region at residues 26–547, a transmembrane segment at 548–568, and a cytoplasmic region at 569–976 (UniProt P21709 topology). HPA also describes cytoplasmic and membranous tissue staining, but rates its tissue IHC reliability Uncertain (HPA tissue IHC). Review cell borders at high magnification and record cytoplasmic colour separately, with the same scoring rule across sections (standard IHC practice). Compare the observed cell population with annotated examples, such as glial cells in cerebral cortex and glandular cells in appendix (HPA tissue IHC). Diffuse colour without discernible cell boundaries warrants background controls before biological interpretation (standard IHC practice).
How can epitope position change the meaning of EPHA1 staining?
Ask for the catalog antibody’s mapped immunogen or epitope before interpreting compartment differences, because this payload does not locate its binding site (supplied antibody evidence). EPHA1 has 3 isoforms, an extracellular region at residues 26–547, and a cytoplasmic region at 569–976 (UniProt P21709). Its reported glycosylation site is residue 414, while reported phosphorylation sites include cytoplasmic residues 599, 605, and 781 (UniProt P21709). These positions guide epitope questions but do not establish which modifications affect this antibody (UniProt P21709; supplied antibody evidence). If isoform specificity matters, compare epitope mapping with the isoform sequences before assigning staining to one isoform (standard assay interpretation).
How should I examine EPHA1 localisation in a multiplex IF experiment?
For the separate IF/ICC workflow, pair EPHA1 with a validated marker of the cell population under study; a glial marker is relevant when examining the brain cell populations annotated by HPA (HPA tissue IHC; standard IF practice). Choose a spectrally separated far-red channel for a weak EPHA1 signal when tissue autofluorescence is troublesome, and inspect single-stain controls before interpreting overlap (standard IF practice). Select permeabilisation after confirming the antibody epitope: extracellular residues 26–547 face outward, whereas residues 569–976 are cytoplasmic (UniProt P21709 topology). The supplied PA1573 caption documents IHC(P) in rat brain tissue, not IF/ICC performance or fixation conditions (PA1573 tissue-IHC caption). Treat apparent colocalisation as spatial evidence requiring antibody and channel controls (standard IF practice).
What controls help identify nonspecific EPHA1 chromogenic staining?
Run a no-primary control through the same secondary reagent, peroxidase block, chromogen, and counterstain sequence to assess detection background (standard IHC practice). Block endogenous peroxidase before DAB development and compare the control with the stained section under identical imaging conditions (standard IHC practice). Titrate the IHC-validated antibody only after checking that retrieval follows citrate pH 6.0, 95–98 °C for 20 min (page retrieval setting; standard IHC practice). Treat colour over folds, damaged edges, or necrotic areas cautiously, and compare intact cell borders because EPHA1 is a membrane receptor (standard IHC practice; UniProt P21709). HPA reports smooth muscle cells as not detected, but its tissue IHC reliability is Uncertain (HPA tissue IHC).
How can I quantify EPHA1 IHC across sections without mixing compartments? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored cell population and membrane versus cytoplasmic compartments before measuring chromogenic signal (standard IHC practice; UniProt P21709 topology). Report the percentage of positive cells and an intensity-weighted H-score separately for each compartment, using the same thresholds and counterstain review across sections (standard IHC practice). If counting discrete positive cells, report density per mm² of viable tissue and document the analysed area (standard IHC practice). Normalise positive-cell counts to the number of eligible cells in the specified population, or density to viable tissue area, while excluding folds and necrosis (standard IHC practice). Annotate cell types because HPA reports differing tissue cell patterns and rates its IHC reliability Uncertain (HPA tissue IHC).
When does EPHA1 colour support a true positive rather than artefact?
A plausible positive shows reproducible cell-associated colour with a clear membrane component, consistent with EPHA1’s cell membrane assignment (UniProt P21709). Cytoplasmic colour can be recorded, since HPA describes cytoplasmic and membranous tissue staining, but that resource rates its IHC reliability Uncertain (HPA tissue IHC). Check whether the stained cells match the tissue annotation under review; HPA reports glial cells in cerebral cortex and smooth muscle cells as not detected (HPA tissue IHC). Discount isolated colour at section edges or necrosis, and investigate colour persisting in a no-primary control as possible detection background (standard IHC practice). Interpret carcinoma staining cautiously because EPHA1 overexpression in several carcinomas does not validate any individual IHC result (UniProt P21709).
Boster reagents
Best EPHA1 / Ephrin type-A receptor 1 IHC Antibodies
Two anti-EPHA1 antibodies have IHC figures from rat brain, human colon cancer, and human tonsil (catalog: IHC image captions); catalog reactivity covers human, mouse, and rat (catalog: reactivity lists).
Real IHC data
Anti-Eph receptor A1/EPHA1 Antibody ®
Cat # PA1573
Real IHC data
Anti-EPHA1 (Phospho-Tyr605) Antibody
Cat # A03024Y605-1
PA1573 is listed for IHC and shows rat brain and human colon cancer tissue IHC(P) (catalog: PA1573 applications and IHC captions). A03024Y605-1 is listed for IHC and IF, with an IHC figure from a paraffin-embedded human tonsil section (catalog: A03024Y605-1 applications and IHC caption).
Which to pick: For tissue IHC targeting EPHA1, choose PA1573; its captions show rat brain and human colon cancer IHC(P), and its listed reactivity includes human, mouse, and rat (catalog: PA1573 IHC captions and reactivity). For phospho-Tyr605 IHC or IF, choose A03024Y605-1; it is listed as polyclonal, reacts with human and mouse, and has a paraffin-section IHC figure from human tonsil (catalog: A03024Y605-1 title, dilution_raw, reactivity, applications, and IHC caption). The figure captions support paraffin-section use but do not report a fixative; ICC is not separately listed (catalog: IHC captions and applications).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P21709 (EPHA1_HUMAN, Ephrin type-A receptor 1).
- Human Protein Atlas. EPHA1 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. EPHA1 subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. EPHA1 antibody validation summary (1 antibodies).
- Association Between EphA1 and Tumor Microenvironment in Gastric Carcinoma and its Clinical Significance. Medical science monitor : international medical journal of experimental and clinical research 2020 — PMC7133419.
- Epigenetic silencing of EphA1 expression in colorectal cancer is correlated with poor survival. British journal of cancer 2009 — PMC2670002.
- Ephrin (Eph) receptor A1, A4, A5 and A7 expression in human non-small cell lung carcinoma: associations with clinicopathological parameters, tumor proliferative capacity and patients' survival. BMC clinical pathology 2014 — PMC4234387.
- Ephrin Receptors (Ephs) Expression in Thymic Epithelial Tumors: Prognostic Implications and Future Therapeutic Approaches. Diagnostics (Basel, Switzerland) 2021 — PMC8700455.
- PubMed PMID:2825356 — UniProt-cited evidence.
- PubMed PMID:10369740 — UniProt-cited evidence.
- PubMed PMID:18593464 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for EPHA1 (Ephrin type-A receptor 1). Expected localisation Membranous and cytoplasmic in most tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: PA1573 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Smooth muscle. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet PA1573); 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-EPHA1, 0.5-1μg/ml (datasheet PA1573); 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 PA1573); Caveat: Phospho-Tyr605 staining depends on site phosphorylation (UniProt; catalog A03024Y605-1); Isoform / epitope: 3 isoforms; map epitopes to extracellular or cytoplasmic regions (UniProt); Regulation: Overexpressed in several carcinomas (UniProt). Evidence: UniProt P21709, HPA tissue IHC · Uncertain, HPA ICC-IF · Membrane, Datasheet PA1573, PMC7133419, PMC4234387. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/EPHA1/immunohistochemistry-ihc-guide.