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EPHB4 / Ephrin type-B receptor 4 · IHC design guide
Design Immunohistochemistry for EPHB4
Plan EPHB4 staining in paraffin sections using colon glandular cells or liver hepatocytes as positive tissue examples (HPA tissue IHC). The catalog antibody’s IHC dilution is 1:100–1:300 (datasheet A00690); interpret cytoplasmic staining in light of the uncertain tissue evidence (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
EPHB4 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 | Tissue IHC: cytoplasmic (HPA tissue IHC); membrane protein (UniProt) | |
| Staining pattern | Glandular cells and hepatocytes show cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A00690) | |
| Positive control | Appendix+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 consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Antibody staining has low consistency with RNA (HPA tissue IHC) | |
| Regulation | Expression regulation unreported (UniProt) | |
| Isoform / epitope | 4 isoforms; map epitopes to extracellular or cytoplasmic regions (UniProt) |
Section 1
Recommended EPHB4 IHC & IF Protocols
The catalog antibody’s IHC-P protocol uses Tris-EDTA pH 9.0 retrieval (datasheet A00690). Published EPHB4 IHC protocols cover prostate, colon, and oral carcinoma samples (PMC1266025; PMC64642; PMC12044661).
| Sample | Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A00690) |
| Fixation | Image fixative and duration unreported (datasheet A00690); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Tris-EDTA pH 9.0 (datasheet A00690); 20 min, 95–100 °C (standard) |
| 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-EPHB4, 1:100-1:300 (datasheet A00690) |
| 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 | EPHB4-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 heat retrieval for the catalog antibody (datasheet A00690). Use each paper’s stated conditions when reproducing its staining (PMC1266025; PMC64642; PMC12044661).
Section 2
What Is the Expected EPHB4 Staining Pattern?
EPHB4 is a single-pass cell-membrane receptor with an extracellular region and a cytoplasmic kinase region (UniProt P54760 topology). In paraffin-section IHC, assess staining in the cell types HPA reports as positive, including intestinal glandular cells and hepatocytes (HPA: High). HPA also describes cytoplasmic staining across several tissues, but rates its tissue IHC profile Uncertain because antibody staining and RNA expression show low consistency (HPA: Uncertain).
| Membrane-associated staining, with possible cytoplasmic staining, in intestinal glandular cells or hepatocytes. | This is compatible with EPHB4's membrane topology and HPA's reported cytoplasmic IHC profile (UniProt P54760 topology; HPA: High in glandular cells and hepatocytes). Record membrane and cytoplasmic signal separately; HPA's Uncertain rating limits how confidently either pattern identifies EPHB4 (HPA: Uncertain). |
| Strong, isolated nuclear staining with little membrane or cytoplasmic signal. | Nuclear localisation is not the expected pattern for this receptor (UniProt P54760: cell membrane; HPA: cytoplasmic IHC profile). Treat an isolated nuclear pattern as suspect and compare it with a no-primary control and staining from an independent antibody (general IHC practice). |
| Prominent staining in adipocytes or lymph-node non-germinal-center cells. | HPA reports EPHB4 as Not detected in these cells, so prominent staining warrants a specificity check (HPA: Not detected). Possible contributors include cross-reactivity or endogenous detection activity; controls can help distinguish them (general IHC practice). HPA's Uncertain rating prevents a definitive negative-tissue claim (HPA: Uncertain). |
| Diffuse color across cells and surrounding tissue without clear cellular boundaries. | This is difficult to score as receptor localisation and may reflect nonspecific background or detection chemistry (general IHC practice). Compare with a no-primary control before interpreting weak cytoplasmic color, particularly because HPA reports a cytoplasmic profile with Uncertain reliability (HPA: Uncertain). |
| No signal in a colon glandular-cell section run as a positive reference. | HPA reports High staining in colon glandular cells, making them a useful comparison, but its overall IHC reliability is Uncertain (HPA: High in colon glandular cells; HPA: Uncertain). Check section quality and assay controls before concluding that the sample lacks EPHB4 (general IHC practice). |
💡Expected EPHB4 appearanceA plausible positive is discernible membrane-associated staining, potentially accompanied by cytoplasmic staining, in glandular cells or hepatocytes reported High by HPA; isolated nuclear or diffuse noncellular color is suspect (UniProt P54760 topology; HPA: cytoplasmic profile, High in these cells, Uncertain; general IHC practice).
| Cellular compartment | EPHB4 spans the membrane at residues 540–560, with extracellular residues 16–539 and cytoplasmic residues 561–987 (UniProt P54760 topology). HPA describes cytoplasmic tissue IHC staining (HPA: cytoplasmic profile). Score membrane and cytoplasmic staining explicitly; neither source establishes that every positive section must show a crisp membrane rim. |
| Choice of comparison tissue | HPA reports High staining in appendix, colon, duodenum and small-intestine glandular cells and in hepatocytes; adipocytes and skeletal-muscle myocytes are Not detected (HPA: tissue IHC). These are observed cell-specific patterns, not guaranteed positive or negative controls, because the tissue profile is rated Uncertain (HPA: Uncertain). |
| Antibody validation | The listed antibody, CAB013537, has an Uncertain IHC validation status (HPA: CAB013537 IHC Uncertain). HPA also reports low consistency between antibody staining and RNA expression (HPA: Uncertain). Interpret unexpected patterns with controls and, where feasible, an independently validated antibody (general IHC practice). |
| Epitope and isoform scope | The receptor has an extracellular region, a cytoplasmic region and 4 listed isoforms (UniProt P54760 topology; UniProt P54760 isoforms). The supplied record does not locate the antibody epitope or establish which isoforms it detects; consult the antibody's documented epitope before attributing compartment differences to an isoform (general IHC practice). |
| ICC/IF Q: Is its localisation established here? | HPA's subcellular summary says Membrane, but supplies no main-location designation or ICC/IF image-bearing cell lines; CAB013537 has no listed ICC validation. Use the separate IF/ICC guide for that application; these records do not establish an IF staining pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected glandular-cell signal is absent. | The IHC run may have failed, or the sampled cells may differ from HPA's reported High glandular-cell populations (HPA: High in colon and small intestine; general IHC practice). | Confirm the intended cells are present, inspect the counterstain, and compare a concurrently processed reference section and assay controls (general IHC practice). Do not treat HPA's Uncertain profile as proof that every section must stain (HPA: Uncertain). |
| Only nuclei stain strongly. | An isolated nuclear pattern conflicts with UniProt's membrane assignment and HPA's cytoplasmic IHC description (UniProt P54760: cell membrane; HPA: cytoplasmic profile). | Review a no-primary control and compare another antibody or control tissue before scoring it as EPHB4 (general IHC practice). |
| Adipocytes or lymph-node non-germinal-center cells stain strongly. | These cells are reported Not detected by HPA; cross-reactivity or endogenous detection activity is possible (HPA: Not detected; general IHC practice). | Compare no-primary and detection controls and check whether expected positive cells stain in the same run (general IHC practice). Treat the mismatch as a specificity concern, given HPA's Uncertain rating (HPA: Uncertain). |
| Brown color is diffuse across the section. | Background from antibody binding or chromogenic detection can obscure cell boundaries (general IHC practice). | Compare no-primary controls, review blocking and washes, and adjust the detection workflow if background persists (general IHC practice). Score only signal that can be assigned to cells and compartments. |
| Cytoplasmic staining appears without a sharp membrane rim. | HPA describes cytoplasmic tissue IHC staining, while UniProt assigns EPHB4 to the cell membrane; the supplied sources do not resolve that difference (HPA: cytoplasmic profile; UniProt P54760: cell membrane). | Record the compartment as observed and compare controls and an independent antibody before calling it specific (general IHC practice). Report HPA's Uncertain reliability alongside the result (HPA: Uncertain). |
| Staining differs among sections or sampled tissues. | HPA reports different cell-specific levels, including High glandular-cell staining and Not detected adipocytes; its overall IHC profile is Uncertain (HPA: tissue IHC; HPA: Uncertain). | Compare like cell types, section quality and concurrent controls before interpreting a biological difference (general IHC practice). Avoid assigning a change in EPHB4 abundance from staining intensity alone. |
Experiment evidence
IHC & IF images showing EPHB4 staining
A00690 · IHCImmunohistochemical analysis of paraffin-embedded human tonsil. 1, Antibody was diluted at 1:200 (4° overnight). 2, Tris-EDTA, pH9.0 was use
A00690-2 · IHCImmunohistochemistry (IHC) analyzes of EphB4 (E601) pAb in paraffin-embedded human brain tissue.
A00690-2 · IFImmunofluorescence analysis of MCF-7 cells using EphB4 (E601) pAb at dilution of 1:200 (40x lens).
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000196411-EPHB4/tissue/cerebral+cortex#imid_3372184
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000196411-EPHB4/tissue/colon#imid_3372094
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000196411-EPHB4/tissue/liver#imid_3372095
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000196411-EPHB4/tissue/kidney#imid_3372112
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for EPHB4 IHC & IF
🧪Run colon first and look for staining in glandular cells (HPA: High in colon glandular cells). Use adipose tissue as the negative comparator (HPA: Not detected in adipocytes); on the colon slide, assess background in adjacent nonglandular cells, but verify their EPHB4 status before treating them as an internal negative (standard IHC practice).
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for EPHB4; 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; an isotype control matched to the primary antibody’s host species and clonality; and, where available, an EPHB4 knockout or epitope-matched peptide-block control (standard IHC practice). For chromogenic colon IHC, quench endogenous peroxidase and check inflammatory-cell staining against the controls (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A00690 paraffin-section caption does not state a fixative (datasheet: fixative not stated). That caption uses Tris-EDTA at pH 9.0 for retrieval and a 1:200 primary dilution, but it does not establish a retrieval requirement for colon (datasheet: tonsil IHC caption). The supplied evidence does not establish that frozen sections or IF are easier; in colon, assess glandular staining against mucin and inflammatory-cell background (HPA: High in colon glandular cells; standard IHC practice).
HPA tissue IHC evidence for EPHB4
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — 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 |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Liver | Hepatocytes | High | Protein (IHC) | HPA → |
| Small intestine | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Lymph node | Non-germinal center cells | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced EPHB4 IHC Tips
Use the paraffin-section caption as the starting protocol, then judge EPHB4 staining by compartment, cell type, and controls (datasheet A00690; UniProt P54760 topology; HPA tissue IHC).
How should I optimize retrieval if EPHB4 staining is weak in paraffin sections?
Start with heat-mediated Tris-EDTA retrieval at pH 9.0 for this antibody (datasheet A00690). The selected paraffin-section image used that buffer and a 1:200 primary dilution overnight at 4°C (datasheet A00690). If staining remains weak, compare a shorter and longer heating interval on adjacent sections while keeping antibody dilution, detection, and imaging conditions constant (standard IHC practice). Review whether signal appears at cell borders, since EPHB4 is a membrane receptor with an extracellular region at residues 16–539 (UniProt P54760 topology). Include a no-primary control when judging any gain in signal (standard IHC practice).
Can I adjust fixation to recover weak EPHB4 staining?
The selected EPHB4 image documents paraffin-embedded tonsil, but does not report its fixative or fixation duration (datasheet A00690). Target-specific sensitivity to fixation is therefore unknown; the tissue staining pattern and receptor topology do not establish it (datasheet A00690; HPA tissue IHC; UniProt P54760 topology). When comparing fixation conditions, process matched specimens through the same Tris-EDTA pH 9.0 retrieval and chromogenic detection steps (datasheet A00690; standard IHC practice). Record fixation and processing times for each specimen, and assess cell borders, tissue preservation, and no-primary background together (standard IHC practice). Avoid assigning weak staining to fixation alone without a controlled comparison (standard IHC practice).
Should EPHB4 appear at the membrane or in the cytoplasm?
Expect a membrane-associated pattern because EPHB4 has one transmembrane segment at residues 540–560 (UniProt P54760 topology). The HPA tissue profile also describes cytoplasmic staining across several tissues, but rates its antibody staining versus RNA consistency as uncertain (HPA tissue IHC). In chromogenic sections, inspect cell borders at high magnification and document cytoplasmic signal separately rather than treating both patterns as equivalent (UniProt P54760 topology; HPA tissue IHC; standard IHC practice). Compare staining in a reported high-expression compartment, such as colon glandular cells, with a no-primary section (HPA: High in colon glandular cells; standard IHC practice). Diffuse staining alone warrants cautious interpretation (HPA tissue IHC).
How do epitope location and isoforms affect this IHC result?
EPHB4 has 4 recorded isoforms, but the supplied antibody caption does not identify its epitope or isoform coverage (UniProt P54760 isoforms; datasheet A00690). Its extracellular region spans residues 16–539, whereas its cytoplasmic region spans 561–987 (UniProt P54760 topology). Glycosylation is recorded at residues 203, 335, and 426, and several phosphorylation sites lie in the cytoplasmic region (UniProt P54760 modifications). Without an epitope map, do not attribute a change in staining to a particular isoform or modification (UniProt P54760 isoforms; datasheet A00690). If epitope information becomes available, check which isoforms retain that sequence before comparing samples (standard IHC practice).
How should I investigate an EPHB4 pattern by multiplex IF?
Use IF as a separate assay and pair EPHB4 with a validated marker for the cell population being examined; the paraffin IHC caption does not validate an IF protocol (datasheet A00690; standard IF practice). Choose fluorophores after measuring tissue autofluorescence and reserve a channel with clear signal separation for the weaker target (standard IF practice). If the antibody recognizes the extracellular region at residues 16–539, test staining with minimal permeabilisation; a cytoplasmic epitope at 561–987 requires access across the membrane (UniProt P54760 topology; standard IF practice). Because this antibody’s epitope is unreported, confirm its location before selecting that condition (datasheet A00690). Include single-stain and no-primary controls to assess bleed-through and background (standard IF practice).
What should I check when EPHB4 staining is widespread or granular?
First compare the stained section with a no-primary control and review endogenous peroxidase blocking, reagent background, and chromogen development (standard IHC practice). The caption specifies a 1:200 primary dilution overnight at 4°C and a 1:200 secondary for 45 minutes at room temperature, providing a documented starting point (datasheet A00690). If background rises with longer development, shorten that step while processing comparison sections together (standard IHC practice). EPHB4 is annotated at the cell membrane, while the HPA reports cytoplasmic tissue staining with uncertain consistency against RNA data (UniProt P54760 subcellular location; HPA tissue IHC). Score granular cytoplasmic deposits cautiously when controls share the pattern (standard IHC practice).
How can I quantify EPHB4 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, and keep membrane-associated and cytoplasmic staining as separate readouts (UniProt P54760 subcellular location; HPA tissue IHC; standard IHC practice). For a defined cell population, record the percentage of positive cells and intensity categories, then calculate an H-score from 0–300 if intensity scoring is reproducible (standard IHC practice). For spatial analyses, report positive-cell density per mm² of viable tissue or a prespecified region, alongside the number of cells assessed (standard IHC practice). Normalize comparisons to the same cell population or viable tissue area, rather than total section area when composition differs (standard IHC practice). Apply identical staining, imaging, and threshold settings across groups (standard IHC practice).
How do I distinguish credible EPHB4 staining from artefact?
Prioritize reproducible cell-border staining in intact cells because EPHB4 is a membrane receptor (UniProt P54760 subcellular location and topology). Check that the stained cell population fits the question: HPA reports high signal in colon glandular cells but no detected signal in skeletal-muscle myocytes, with uncertain overall staining-to-RNA consistency (HPA tissue IHC). Inspect section edges and necrotic regions for localized artefacts, and use a no-primary control to assess nonspecific deposition (standard IHC practice). Endogenous enzyme activity can also create chromogenic signal, so evaluate the peroxidase block and detection controls (standard IHC practice). Treat cytoplasmic-only staining as provisional until independent controls support it (HPA tissue IHC; standard IHC practice).
Boster reagents
Best EPHB4 / Ephrin type-B receptor 4 IHC Antibodies
A00690 and A00690-2 have paraffin-section IHC images (catalog IHC captions); both list human and mouse reactivity (catalog reactivity), while A00690-2 also lists rat reactivity and has an IF image (catalog reactivity/IF caption).
Real IHC data
Anti-Ephrin type-B receptor 4 EphB4 Antibody
Cat # A00690
Real IHC data
Anti-EphB4 (E601) Antibody
Cat # A00690-2
A00690 shows IHC on paraffin-embedded human tonsil and lists human and mouse reactivity (A00690 IHC caption/catalog reactivity). A00690-2 shows IHC on paraffin-embedded human brain and IF in MCF-7 cells, and lists human, mouse and rat reactivity (A00690-2 IHC and IF captions/catalog reactivity).
Which to pick: For tissue IHC, choose A00690 if its human tonsil example with 1:200 primary dilution and Tris-EDTA retrieval at pH 9.0 fits your plan; A00690-2 has a paraffin-embedded human brain example (respective IHC captions). For IF, A00690-2 has an MCF-7 image at 1:200; for ICC, A00690-3 lists ICC as an application but provides no ICC image (A00690-2 IF caption; A00690-3 catalog applications/image captions). For rat samples, A00690-2 and A00690-3 list rat reactivity; the rendered antibodies have no clone reported, and neither IHC caption reports a fixative (catalog reactivity/clone fields; IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P54760 (EPHB4_HUMAN, Ephrin type-B receptor 4).
- Human Protein Atlas. EPHB4 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. EPHB4 subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. EPHB4 antibody validation summary (1 antibodies).
- EphB4/ TNFR2/ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation. BMC molecular and cell biology 2020 — PMC7164363.
- Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma. BMC cancer 2005 — PMC1266025.
- Receptor protein tyrosine kinase EphB4 is up-regulated in colon cancer. BMC molecular biology 2001 — PMC64642.
- Feasibility of Intratumoral Administration With EPHB4-CAR-T Cells for the Treatment of Oral Squamous Cell Carcinoma. Cancer science 2025 — PMC12044661.
- PubMed PMID:8188704 — UniProt-cited evidence.
- PubMed PMID:11239002 — UniProt-cited evidence.
- PubMed PMID:18593464 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for EPHB4 (Ephrin type-B receptor 4). Expected localisation Tissue IHC: cytoplasmic (HPA tissue IHC); membrane protein (UniProt). Antigen retrieval: Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A00690). Validated IHC antibody: A00690 at 1:100-1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A00690); 2. Fixation: Image fixative and duration unreported (datasheet A00690); 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: Tris-EDTA pH 9.0 (datasheet A00690); 20 min, 95–100 °C (standard); 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-EPHB4, 1:100-1:300 (datasheet A00690); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Antibody staining has low consistency with RNA (HPA tissue IHC); Isoform / epitope: 4 isoforms; map epitopes to extracellular or cytoplasmic regions (UniProt); Regulation: Expression regulation unreported (UniProt). Evidence: UniProt P54760, HPA tissue IHC · Uncertain, HPA ICC-IF · Membrane, Datasheet A00690, PMC1266025, PMC64642. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/EPHB4/immunohistochemistry-ihc-guide.