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EFNA5 / Ephrin-A5 · IHC design guide
Design Immunohistochemistry for EFNA5
Plan chromogenic EFNA5 IHC in paraffin sections using the IHC-validated antibody at 1:50–1:200 as a starting range (datasheet: A06229). Compare staining in neuronal or glandular cells with HPA tissues where EFNA5 was not detected (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
EFNA5 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 tissue staining (HPA tissue IHC) | |
| Staining pattern | Neuronal and glandular cells show membranous and cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Caudate+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 formalin fixation consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | ADAM10 can shed the extracellular domain, altering surface signal (UniProt) | |
| Regulation | Expression regulation is not annotated (UniProt) | |
| Isoform / epitope | No isoforms annotated; mature chain excludes propeptide 204–228 (UniProt) |
Section 1
Recommended EFNA5 IHC & IF Protocols
The catalog antibody’s IHC protocol is accompanied by published EFNA5 protocols for mouse retina, hepatoma tissue arrays, and renal carcinoma arrays (PMC7569469; PMC11489415; PMC8870418).
| Sample | Paraffin-embedded human brain tissue; fixative not specified (datasheet A06229) |
| Fixation | Image fixative and duration unreported (datasheet A06229); 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-EFNA5, 1:50-1:200 (datasheet A06229) |
| 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 | EFNA5-positive staining in neuronal cells of caudate (HPA tissue IHC: High). HPA tissue profile: Membranous and cytoplasmic expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval rule); the mouse retina study used 95 °C for 10 min (PMC7569469).
Section 2
What Is the Expected EFNA5 Staining Pattern?
EFNA5 is a GPI-anchored cell-surface ligand with no transmembrane segment and can occupy caveolae-like membrane microdomains (UniProt P52803 topology). Expect membranous and cytoplasmic staining in several tissues, including neuronal and glandular cells (HPA tissue IHC). HPA rates its tissue staining Approved but reports low consistency with RNA expression; interpret tissue intensity with that caveat (HPA tissue IHC).
| Membranous staining, with some cytoplasmic staining, in caudate or cerebral cortex neuronal cells. | This matches the reported compartments and High neuronal staining (HPA tissue IHC). Cell-surface signal is consistent with GPI-anchored EFNA5 (UniProt P52803 topology). Assess the named cells rather than treating all tissue elements as equivalent (HPA tissue IHC). |
| Predominantly nuclear staining, with little signal at the membrane or in the cytoplasm. | A nuclear-dominant pattern does not match HPA tissue IHC or the reported cell-membrane location (HPA tissue IHC; UniProt P52803 topology). Treat it as a possible staining artefact and compare it with the expected compartments and controls (general IHC practice). |
| Strong staining in adipocytes or skeletal-muscle myocytes instead of the expected positive cells. | HPA reports those cell types as Not detected (HPA tissue IHC). Consider cross-reactivity or endogenous chromogenic activity and review detection controls (general IHC practice). An unexpected positive alone does not establish EFNA5 expression, particularly given HPA's low staining–RNA consistency (HPA tissue IHC). |
| Diffuse color coats the section without clear cellular or compartment boundaries. | That appearance cannot support a cell-specific EFNA5 call (general IHC practice). Check background in a no-primary control, then assess blocking, washes, detection chemistry and chromogen development as general IHC variables (general IHC practice). |
| No staining appears in caudate neuronal cells or duodenal glandular cells. | Both are listed as High in HPA tissue IHC, so a blank positive-control section calls for a workflow check (HPA tissue IHC; general IHC practice). HPA's Approved rating and low staining–RNA consistency mean one negative section cannot, by itself, settle EFNA5 absence (HPA tissue IHC). |
💡Expected EFNA5 appearanceCall a result positive when identifiable neuronal or glandular cells show clear membranous staining, with possible cytoplasmic staining, in an HPA High setting; nuclear-dominant color or diffuse background is a suspect pattern (HPA tissue IHC; UniProt P52803 topology; general IHC practice).
| Membrane topology | EFNA5 is a cell-surface GPI-bound ligand, lacks a transmembrane segment and occupies discrete caveolae-like microdomains (UniProt P52803 topology and function). Judge membrane localization at cell borders; an entirely nuclear pattern conflicts with that annotation (UniProt P52803 topology). |
| Processing and shedding | UniProt lists a signal peptide at residues 1–20, a mature chain at 21–203, and ADAM10-mediated extracellular-domain shedding (UniProt P52803 processing and subunit). These features make epitope location relevant to interpretation, but the payload does not locate the IHC antibody's epitope or establish a specific staining change. |
| Tissue choice and evidence strength | HPA lists High staining in hippocampal neuronal cells, gallbladder glandular cells and cardiomyocytes, among others; adipocytes and bone-marrow hematopoietic cells are Not detected (HPA tissue IHC). HPA also reports low tissue RNA specificity and low staining–RNA consistency, so compare the named cell populations rather than assuming whole-tissue uniformity (HPA tissue IHC). |
| Antibody validation | The HPA antibody CAB013282 has an Approved IHC rating, while HPA054047 has no IHC rating in the supplied record (HPA antibodies). HPA's tissue profile is also Approved with low staining–RNA consistency (HPA tissue IHC). Neither rating establishes that every observed chromogenic signal is EFNA5. |
| IF/ICC Q: Should its image match the IHC slide? | A: HPA reports approved vesicle and cytosol localization in ICC-IF, whereas tissue IHC is described as membranous and cytoplasmic (HPA subcellular ICC-IF; HPA tissue IHC). The modalities and rated antibodies differ (HPA antibodies); use the separate IF/ICC guide for that application. |
| Situation | Likely cause | Next action |
|---|---|---|
| A known-positive cell population is blank. | A weak or failed IHC run is possible; HPA lists caudate neuronal cells and duodenal glandular cells as High (HPA tissue IHC; general IHC practice). | Confirm the positive-control section, primary-antibody step and detection reagents; review antigen-retrieval conditions as a general IHC workflow check (general IHC practice). No EFNA5-specific retrieval or fixation sensitivity is established by these sources. |
| Color is uniform across cells and empty spaces. | Diffuse background can arise from nonspecific detection or excess chromogen development (general IHC practice). It does not reproduce HPA's cell-associated membranous and cytoplasmic profile (HPA tissue IHC). | Inspect a no-primary control and reassess blocking, washing and development time (general IHC practice). Score EFNA5 only where cellular localization remains interpretable (HPA tissue IHC; general IHC practice). |
| The strongest signal is nuclear. | EFNA5 is annotated at the cell membrane, including caveolae-like microdomains, while HPA tissue staining is membranous and cytoplasmic (UniProt P52803 topology; HPA tissue IHC). | Flag the nuclear pattern as suspect; compare it with a positive tissue and detection controls before interpreting it as EFNA5 (HPA tissue IHC; general IHC practice). |
| An HPA Not detected cell type stains strongly. | For adipocytes, skeletal-muscle myocytes or bone-marrow hematopoietic cells, the result conflicts with the listed HPA cell-type pattern (HPA tissue IHC). Cross-reactivity or endogenous detection activity is possible (general IHC practice). | Check the no-primary control and the identity of the stained cells; compare with an HPA High population before calling the signal specific (HPA tissue IHC; general IHC practice). |
| Membrane and cytoplasmic signals vary within an otherwise positive tissue. | HPA describes both compartments, while UniProt places EFNA5 at the surface and reports extracellular-domain shedding (HPA tissue IHC; UniProt P52803 topology and subunit). The supplied evidence does not assign a mechanism to each stained cell. | Record the two compartments separately and compare the same named cell type across sections; avoid inferring shedding from the IHC pattern alone (HPA tissue IHC; UniProt P52803 subunit; general IHC practice). |
| An IF/ICC image appears more vesicular than the IHC section. | HPA's approved ICC-IF localization is vesicles and cytosol; its tissue IHC description is membranous and cytoplasmic (HPA subcellular ICC-IF; HPA tissue IHC). | Interpret each image against its own application-specific reference and antibody rating (HPA antibodies; HPA tissue IHC; HPA subcellular ICC-IF). Use the separate IF/ICC guide for IF/ICC decisions. |
Experiment evidence
IHC & IF images showing EFNA5 staining
A06229 · IHCImmunohistochemistry (IHC) analyzes of Ephrin-A5 (P63) pAb in paraffin-embedded human brain tissue.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000184349-EFNA5/tissue/cerebral+cortex#imid_3688168
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000184349-EFNA5/tissue/colon#imid_3688094
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000184349-EFNA5/tissue/liver#imid_3688087
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000184349-EFNA5/tissue/kidney#imid_3688088
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA054047; green = EFNA5, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA054047; green = EFNA5, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for EFNA5 IHC & IF
🧪Run caudate first: neuronal cells should stain strongly (HPA: High in caudate neuronal cells). Use skeletal muscle myocytes as a negative tissue (HPA: Not detected in skeletal muscle myocytes); cells without specific staining on the caudate slide should show only background, but the payload does not identify a validated internal negative cell type.
Positive control tissue: Caudate (Neuronal cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show EFNA5 in A-431, U-251MG, U2OS, NIH 3T3, with annotated localisation: Vesicles (approved), Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, a host-matched nonimmune polyclonal immunoglobulin control for the catalog pAb (selected A06229 caption: pAb), and EFNA5 knockout material where available as a biological negative. For chromogenic IHC, quench endogenous peroxidase and inspect the brain section for pigment that could be mistaken for signal (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A06229 paraffin-section caption does not state the fixative (selected A06229 caption: paraffin-embedded human brain tissue; fixative unreported). EFNA5-specific antigen-retrieval dependence is unreported, so assess retrieval conditions against the positive and negative tissues; the supplied evidence does not establish whether frozen sections or IF are easier. In brain, distinguish pigment from chromogenic staining (standard IHC practice), and interpret IF localisation cautiously because HPA reports vesicles and cytosol while UniProt places EFNA5 at the cell membrane (HPA: approved vesicle and cytosol localisation; UniProt P52803: cell membrane).
HPA tissue IHC evidence for EFNA5
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 |
|---|---|---|---|---|
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Epididymis | 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 → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Ovary | Ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced EFNA5 IHC Tips
These questions address EFNA5 staining in paraffin sections with chromogenic detection; the immunofluorescence entry covers the secondary application.
How should I adjust retrieval when EFNA5 staining is weak?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval specification). After cooling, keep section thickness, detection chemistry and the catalog antibody dilution constant while comparing retrieval runs (general IHC practice). Use a neuronal cell region in cerebral cortex as a reference for expected staining (HPA: High in cerebral cortex neuronal cells). If staining remains weak, test a different retrieval buffer or pH on adjacent sections as a fallback, while monitoring tissue damage (general IHC practice). Record the tested conditions with each image so changes in staining can be attributed to retrieval (general IHC practice).
Can fixation explain inconsistent EFNA5 staining across paraffin blocks?
The selected catalog image establishes staining in paraffin embedded human brain tissue, but its caption does not report the fixative (A06229 caption). EFNA5 sensitivity to any particular fixative or fixation duration is therefore unknown from the supplied target specific evidence (A06229 caption). Compare blocks with documented processing histories, using adjacent sections and the same citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval specification; general IHC practice). Include a reference section in each staining run and inspect morphology alongside signal intensity (general IHC practice). Treat a block dependent difference as a processing question until matched controls support a biological interpretation (general IHC practice).
Should EFNA5 appear at cell borders or in the cytoplasm?
Prioritize membrane associated staining because EFNA5 is a cell surface GPI bound ligand that occupies caveolae like membrane microdomains (UniProt P52803 function and subcellular annotation). Cytoplasmic staining can occur in tissue sections (HPA: membranous and cytoplasmic tissue expression), and vesicles and cytosol are approved IF locations (HPA subcellular). Examine borders, puncta and diffuse cytoplasm separately at matched magnification, with the same hematoxylin counterstain and exposure settings (general IHC practice). For suspected neuronal signal, compare stained cells with tissue morphology in cerebral cortex or hippocampus (HPA: High in neuronal cells of both tissues; general IHC practice). Do not equate diffuse chromogen alone with surface ligand abundance (UniProt P52803 topology; general IHC practice).
Could epitope placement explain different EFNA5 staining patterns?
Check the catalog antibody immunogen before assigning a staining pattern to a specific EFNA5 region (general IHC practice). The annotated mature chain spans residues 21–203, with a signal peptide at 1–20 and a propeptide at 204–228 (UniProt P52803 processing). The Ephrin receptor binding domain spans 29–162, and a glycosylation site is annotated at residue 37 (UniProt P52803 domains and glycosylation). No isoforms are listed in the supplied record, so an isoform specific explanation lacks support here (UniProt P52803 isoforms). If the antibody epitope is unspecified, compare membrane and cytoplasmic patterns without claiming that chromogenic staining distinguishes intact ligand from processed material (UniProt P52803 topology and processing; general IHC practice).
How can IF clarify EFNA5 localisation alongside neuronal markers?
For the separate IF application, multiplex EFNA5 with a neuronal marker in a region where neuronal cells show high tissue staining, such as cerebral cortex (HPA: High in cerebral cortex neuronal cells; general IF practice). Choose spectrally separated fluorophores and compare an unstained section to assess tissue autofluorescence before selecting the EFNA5 channel (general IF practice). If the antibody recognizes an extracellular epitope, assess surface staining without permeabilisation; if its epitope is intracellular or the question concerns internal pools, test mild permeabilisation on a matched section (UniProt P52803 GPI bound topology; general IF practice). Keep single stain controls for bleed through and interpret vesicular or cytosolic signal alongside the neuronal marker (HPA subcellular: vesicles and cytosol approved; general IF practice).
What should I check when EFNA5 chromogen stains widely?
Run a no primary control through the same secondary reagent and chromogen steps to reveal detection system background (general IHC practice). Include a peroxidase block before chromogenic detection and check whether residual enzyme activity contributes to the DAB signal (general IHC practice). Compare suspected nonspecific staining with adipocytes or skeletal muscle myocytes, where EFNA5 was not detected in the supplied tissue profile (HPA: adipocytes and skeletal muscle myocytes Not detected). Reduce primary antibody concentration only through a documented dilution series, and keep retrieval and development time matched across sections (general IHC practice). Interpret diffuse staining cautiously because tissue expression includes cytoplasmic signal, while EFNA5 is GPI bound at the cell surface (HPA tissue profile; UniProt P52803 topology).
How should I score EFNA5 across tissue regions? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because tissue staining is reported at membranes and in cytoplasm (HPA tissue profile). For chromogenic sections, record the percentage of positive cells and staining intensity, then calculate an H score from intensity weighted percentages if that scale suits the study (general IHC practice). Alternatively, report positive cell density per mm² of viable tissue and keep membrane and cytoplasmic scores separate (general IHC practice). Normalize counts to the number of eligible cells or measured viable area, and use the same segmentation and threshold rules for every section (general IHC practice). Report neuronal regions separately from glandular regions when both are sampled (HPA: High in neuronal and glandular cells; general IHC practice).
When is an EFNA5 positive cell convincing rather than artefactual?
A convincing result follows cell morphology and a plausible compartment: EFNA5 is membrane associated, while tissue IHC also reports cytoplasmic staining (UniProt P52803 subcellular annotation; HPA tissue profile). Check whether signal appears in expected neuronal cells of cerebral cortex or hippocampus, with an adjacent negative control processed in the same run (HPA: High in those neuronal cells; general IHC practice). Treat staining concentrated at section edges, necrotic areas or sites of endogenous enzyme activity as suspect until controls resolve it (general IHC practice). Compare a putative negative population, such as skeletal muscle myocytes, without treating one negative section as proof of antibody specificity (HPA: skeletal muscle myocytes Not detected; general IHC practice). HPA reports low consistency between antibody staining and RNA data, so corroborate biological conclusions independently (HPA reliability description; general IHC practice).
Boster reagents
Best EFNA5 / Ephrin-A5 IHC Antibodies
A06229 has an IHC image from paraffin-embedded human brain tissue (A06229 image caption), lists IF as an application, and lists human, mouse and rat reactivity (A06229 catalog applications/reactivity).
Real IHC data
Anti-Ephrin-A5 (P63) EFNA5 Antibody
Cat # A06229
Only A06229 will render; its own figure shows IHC on paraffin-embedded human brain tissue (A06229 image caption). It lists IHC and IF applications and human, mouse and rat reactivity (A06229 catalog applications/reactivity); no IF figure is supplied (A06229 IF image alts).
Which to pick: Choose A06229 for paraffin-section tissue IHC because its own figure shows human brain tissue processed as paraffin sections (A06229 image caption); the fixative is unreported (A06229 image caption). For IF, A06229 is listed at 1:50–1:200 (A06229 catalog applications/dilution), but no IF image or ICC application is supplied (A06229 catalog IF image alts/applications). For cross-species work, A06229 lists human, mouse and rat reactivity (A06229 catalog reactivity); its IHC figure documents human tissue only (A06229 image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P52803 (EFNA5_HUMAN, Ephrin-A5).
- Human Protein Atlas. EFNA5 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. EFNA5 subcellular location (ICC-IF): Localized to vesicles and cytosol..
- Human Protein Atlas. EFNA5 antibody validation summary (2 antibodies).
- Ephrin-A5 Is Involved in Retinal Neovascularization in a Mouse Model of Oxygen-Induced Retinopathy. BioMed research international 2020 — PMC7569469.
- EFNA5 suppresses cell proliferation and tumor metastasis in hepatoma via epithelial-to-mesenchymal transition. Discover oncology 2024 — PMC11489415.
- c-Met and EPHA7 Receptor Tyrosine Kinases Are Related to Prognosis in Clear Cell Renal Cell Carcinoma: Focusing on the Association with Myoferlin Expression. Cancers 2022 — PMC8870418.
- EphrinA5 Signaling Is Required for the Distinctive Targeting of Raphe Serotonin Neurons in the Forebrain. eNeuro 2017 — PMC5292598.
- PubMed PMID:7748564 — UniProt-cited evidence.
- PubMed PMID:9245480 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for EFNA5 (Ephrin-A5). Expected localisation Membranous and cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A06229 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Caudate (Neuronal cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human brain tissue; fixative not specified (datasheet A06229); 2. Fixation: Image fixative and duration unreported (datasheet A06229); 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-EFNA5, 1:50-1:200 (datasheet A06229); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep formalin fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: ADAM10 can shed the extracellular domain, altering surface signal (UniProt); Isoform / epitope: No isoforms annotated; mature chain excludes propeptide 204–228 (UniProt); Regulation: Expression regulation is not annotated (UniProt). Evidence: UniProt P52803, HPA tissue IHC · Approved, HPA ICC-IF · vesicles and cytosol., Datasheet A06229, PMC7569469, PMC11489415. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/EFNA5/immunohistochemistry-ihc-guide.