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EPN2 / Epsin-2 · IHC design guide
Design Immunohistochemistry for EPN2
Plan paraffin-section EPN2 IHC around medium neuronal staining in caudate, cerebral cortex and hippocampus (HPA tissue IHC). This guide covers a 1:200 starting dilution (datasheet A08001-1), expected cytoplasmic vesicle localisation (UniProt), and specificity controls prompted by presumed off-target staining (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
EPN2 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 vesicles expected (UniProt); IHC compartment unreported (HPA tissue IHC) | |
| Staining pattern | Medium neuronal staining in CNS; compartment and texture unreported (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, heat-mediated (datasheet A08001-1) | |
| Positive control | Caudate+2 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). Selected-image fixative and duration unreported (datasheet A08001-1); verify before use. | |
| Caveat | Presumed off-target staining may affect interpretation (HPA tissue IHC) | |
| Regulation | Highest expression in brain (UniProt) | |
| Isoform / epitope | 4 isoforms; no extracellular segment; epitope coverage unknown (UniProt) |
Section 1
Recommended EPN2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet A08001-1) is accompanied by a published EPN2 protocol for paraffin-embedded bladder cancer tissue (PMC12573180).
| Sample | Paraffin-embedded human small intestinal carcinoma tissue; fixative not specified (datasheet A08001-1) |
| Fixation | Image fixative and duration unreported (datasheet A08001-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6.0, 20min (datasheet A08001-1) |
| 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-EPN2, 1:100-1:300 (datasheet A08001-1) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | EPN2-positive staining in neuronal cells of caudate (HPA tissue IHC: Medium). HPA tissue profile: Expression in CNS. No signal in the no-primary control. |
💡Decision noteTry heat-mediated citrate retrieval at pH 6.0 first (datasheet A08001-1); the published EPN2 method does not specify retrieval (PMC12573180).
Section 2
What Is the Expected EPN2 Staining Pattern?
EPN2 should appear mainly as cytoplasmic vesicular staining in neuronal cells of the caudate, cerebral cortex, and hippocampus (UniProt O95208: cytoplasm and clathrin-coated vesicles; HPA: Medium in neuronal cells). It has no transmembrane segment (UniProt O95208 topology). Interpret the pattern cautiously: HPA rates tissue IHC Approved but reports low consistency with RNA data and presumed off-target staining that was disregarded (HPA: tissue IHC reliability).
| Discrete cytoplasmic puncta in neuronal cells, with moderate staining in CNS sections. | This fits EPN2’s vesicular location and the observed neuronal pattern in caudate, cortex, and hippocampus (UniProt O95208: subcellular location; HPA: Medium in neuronal cells). Golgi-region concentration is plausible, but its absence alone does not invalidate an otherwise vesicular pattern (UniProt O95208: Golgi-region note). |
| Predominantly nuclear staining, or a continuous cell-surface rim. | Neither is the expected EPN2 compartment: UniProt places it in cytoplasm and clathrin-coated vesicles and lists no transmembrane segment (UniProt O95208: location and topology). Check compartment assignment and staining controls before scoring such signal as EPN2 (general IHC practice). |
| Strong staining in adipocytes or other cells listed as undetected by HPA. | HPA reports EPN2 as Not detected in adipose-tissue adipocytes and several other sampled cell types (HPA: tissue IHC). Unexpected staining raises concern for cross-reactivity or endogenous detection activity; confirm with appropriate controls. HPA’s Approved rating and low RNA concordance limit how firmly any single tissue can serve as a negative (HPA: reliability; general IHC practice). |
| Uniform haze across cells, stroma, or blank areas instead of localized puncta. | Diffuse background is less consistent with the reported cytoplasmic vesicle pattern (UniProt O95208: location; HPA: vesicles supported by ICC-IF). Review no-primary and detection controls, then optimize blocking, washing, and antibody concentration as general IHC troubleshooting steps (general IHC practice). |
| No staining in neuronal cells of a caudate, cortex, or hippocampus section. | That conflicts with HPA’s Medium neuronal staining in those tissues (HPA: tissue IHC). Check that the relevant cells are present and that the positive control and detection reagents work. A negative section alone cannot establish absent EPN2, particularly given HPA’s stated antibody–RNA inconsistency (HPA: reliability; general IHC practice). |
💡Expected EPN2 appearanceCall a result positive when neuronal cells show Medium cytoplasmic vesicular staining in the sampled CNS regions (HPA: tissue IHC; UniProt O95208: location); nuclear, surface-rim, or widespread diffuse signal needs control-based review (UniProt O95208: topology; general IHC practice).
| Compartment and topology | EPN2 is cytoplasmic, associates with clathrin-coated vesicles, and lacks a transmembrane segment (UniProt O95208: location and topology). Score intracellular puncta in cell context; a membrane-like rim does not match that annotation. |
| Tissue and cell context | HPA records Medium neuronal staining in caudate, cerebral cortex, and hippocampus, but Not detected in cerebellar granular-layer cells (HPA: tissue IHC). Choose and interpret controls at the cell-type level rather than treating all brain regions alike. |
| IHC evidence strength | One listed antibody has IHC Approved status, without an IHC Enhanced designation; HPA also flags low staining–RNA consistency and presumed off-target binding that was disregarded (HPA: antibodies and reliability). Treat unusual staining as provisional until controls support it. |
| Isoforms and assay scope | UniProt lists 4 EPN2 isoforms (UniProt O95208: isoforms). The supplied evidence gives no antibody epitope or isoform-specific IHC pattern, so section staining cannot be assigned to a particular isoform from these records alone. |
| IF/ICC Q&A: what pattern is expected? | Vesicles are the supported main ICC-IF location, with images listed for A-549 and U2OS (HPA: subcellular). The listed ICC Supported antibody differs from the IHC Approved antibody (HPA: antibodies); these records do not establish an IF protocol for paraffin sections. |
| Situation | Likely cause | Next action |
|---|---|---|
| CNS neuronal positive control is blank. | The observed HPA pattern predicts Medium neuronal staining in caudate, cortex, or hippocampus; a blank result may reflect assay failure or sample context (HPA: tissue IHC; general IHC practice). | Verify neuronal cells and section quality, then check the IHC-validated antibody, detection reagents, and positive-control run. Optimize retrieval using assay controls; no EPN2-specific retrieval condition is supplied (general IHC practice). |
| Diffuse color obscures intracellular detail. | The distribution does not resolve the vesicular localization reported for EPN2 (UniProt O95208: location; HPA: subcellular). Excess background can arise in chromogenic IHC (general IHC practice). | Inspect no-primary and detection controls; review blocking, washing, and antibody concentration, then reassess whether distinct cytoplasmic puncta remain (general IHC practice). |
| Signal appears mainly nuclear or at cell borders. | Those compartments conflict with cytoplasmic vesicle localization and the absence of a transmembrane segment (UniProt O95208: location and topology). | Check counterstain boundaries and focus; compare a control section and the expected neuronal-cell pattern before recording the signal as EPN2 (HPA: Medium in neuronal cells; general IHC practice). |
| Adipocytes or other HPA-undetected cells stain strongly. | The staining differs from HPA’s sampled cell-type observations; cross-reactivity or endogenous chromogen-generating activity are possible explanations (HPA: tissue IHC; general IHC practice). | Run no-primary and detection controls and check endogenous enzyme blocking where relevant. Treat the result cautiously because HPA reports low antibody-staining concordance with RNA (HPA: reliability; general IHC practice). |
| All sections, including the no-primary control, develop color. | A no-primary control cannot report EPN2 antibody binding; staining there points toward detection-system background or endogenous activity (general IHC practice). | Check the detection reagents and blocking steps, including endogenous enzyme blocking for the chosen chromogen, before interpreting tissue differences (general IHC practice). |
| A cerebellar granular-layer sample is blank while another CNS sample stains. | This can match the supplied observations: HPA reports Not detected in granular-layer cells but Medium staining in neuronal cells of caudate, cortex, and hippocampus (HPA: tissue IHC). | Score the named cell populations separately and use an HPA-observed positive region to assess assay performance. Do not call the blank granular layer a failed run by itself (HPA: tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing EPN2 staining
A08001-1 · IHCImmunohistochemical analysis of paraffin-embedded human small intestinal carcinoma tissue. 1, primary Antibody was diluted at 1:200 (4° over
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000072134-EPN2/tissue/cerebral+cortex#imid_13770005
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000072134-EPN2/tissue/colon#imid_13773451
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000072134-EPN2/tissue/liver#imid_13773452
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000072134-EPN2/tissue/kidney#imid_13773461
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA053360; green = EPN2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA053360; green = EPN2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for EPN2 IHC & IF
🧪Run caudate first: neuronal cells should stain at a Medium level (HPA: caudate neuronal cells, Medium). Use adipose tissue as the negative comparator because its adipocytes are not detected (HPA: adipose tissue adipocytes, Not detected); non-neuronal cells on the caudate slide should remain near background as an internal check, but their EPN2 status is not specified by the supplied HPA rows.
Positive control tissue: Caudate (Neuronal cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show EPN2 in A-549, U2OS, with annotated localisation: Vesicles (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species and clonality (standard IHC practice); use EPN2 knockout material or a validated peptide block as a biological specificity control (standard IHC practice). For chromogenic detection, block endogenous peroxidase and check for endogenous biotin if using a biotin-based system; for fluorescence, assess tissue autofluorescence (standard IHC/IF practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported in the supplied evidence; the selected A08001-1 paraffin-section caption also leaves the fixative unreported (selected-SKU IHC caption). The caption uses sodium citrate at pH 6.0 for retrieval above 98°C for 20 minutes, but it does not establish that retrieval is required under all conditions (selected-SKU IHC caption). IF/ICC can help assess the expected vesicular pattern in A-549 or U2OS cells (HPA: subcellular vesicles; ICC-IF images in A-549 and U2OS); no supplied evidence establishes that frozen sections are easier or identifies a tissue-specific artefact.
HPA tissue IHC evidence for EPN2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low consistency between antibody staining and RNA expression data. Presumed off target binding observed and disregarded.). 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 | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | Medium | Protein (IHC) | HPA → |
| Hippocampus | Neuronal cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced EPN2 IHC Tips
Troubleshoot EPN2 staining in paraffin sections by checking retrieval, compartment, controls and scoring alongside the reported neuronal staining (HPA tissue IHC).
What retrieval conditions should I try when EPN2 staining is weak?
Use heat-mediated sodium citrate at pH 6.0 for paraffin-section EPN2 IHC (datasheet A08001-1). The selected image used retrieval above 98°C for 20 minutes, followed by primary antibody at 1:200 overnight at 4°C (caption A08001-1). If signal is weak, compare a longer heating interval on adjacent sections while keeping the antibody dilution and detection chemistry fixed (standard IHC practice). Check whether neuronal cytoplasmic staining improves without a parallel rise in diffuse background; neuronal staining is reported in several CNS regions (HPA tissue IHC), and EPN2 is cytoplasmic and vesicle-associated (UniProt O95208). Record cooling conditions and section retention so changes in staining can be attributed to retrieval (standard IHC practice).
How should I assess whether fixation is limiting EPN2 staining?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not report a fixative (caption A08001-1). Record fixative, fixation duration, tissue thickness and processing history for each specimen before comparing EPN2 staining (standard IHC practice). Where matched material is available, stain sections processed under different documented fixation conditions in one run, using the same citrate pH 6.0 retrieval and antibody conditions (datasheet A08001-1; standard IHC practice). Interpret weaker staining only after checking morphology, section integrity and a concurrently processed positive control (standard IHC practice). Neither the reported neuronal pattern nor EPN2's topology establishes a fixation effect (HPA tissue IHC; UniProt O95208).
What cellular staining pattern is plausible for EPN2?
Expect predominantly cytoplasmic staining that may appear punctate, with enrichment near the Golgi region, because EPN2 associates with clathrin-coated vesicles (UniProt O95208). Supported subcellular imaging also localizes EPN2 to vesicles, although those images are from ICC/IF rather than this paraffin assay (HPA subcellular). Inspect neuronal cell bodies at sufficient magnification to distinguish puncta from pigment, precipitate and overlapping cells; neuronal staining is reported in caudate, cerebral cortex and hippocampus (HPA tissue IHC; standard IHC practice). A strong, exclusively nuclear or uniform surface-membrane pattern warrants specificity checks because EPN2 has no transmembrane segment and its reported locations are cytoplasmic and vesicular (UniProt O95208).
Could isoforms or epitope placement explain inconsistent EPN2 staining?
EPN2 has 4 annotated isoforms, so confirm which sequence the antibody immunogen covers before comparing staining across specimens (UniProt O95208; standard IHC practice). The record places the ENTH domain at residues 12–144 and two UIMs at 275–294 and 300–319, but supplies no epitope for this antibody (UniProt O95208; supplied product evidence). Document the immunogen when available and check whether the chosen epitope is shared across isoforms before interpreting a negative stain (standard IHC practice). EPN2 also has annotated methylation and phosphorylation sites; these annotations alone do not establish altered antibody binding in processed sections (UniProt O95208). Compare any discrepant result with an independently validated antibody or orthogonal expression evidence (standard IHC practice).
How can I check the IHC pattern by multiplex immunofluorescence?
For a separate IF/ICC experiment, pair EPN2 with a validated neuronal marker when examining the CNS cell population reported to stain in tissue IHC (HPA tissue IHC; standard IF practice). Select spectrally separated fluorophores and place the weaker signal in a channel with low measured tissue autofluorescence; include single-color and no-primary controls (standard IF practice). EPN2 is cytoplasmic and associated with vesicles, with no transmembrane segment, so use controlled permeabilisation to expose an intracellular epitope while preserving puncta (UniProt O95208; standard IF practice). HPA reports supported vesicular localization in ICC/IF images from A-549 and U2OS cells, which can inform pattern comparison but does not validate this IHC antibody for IF (HPA subcellular; supplied product evidence).
What should I adjust when chromogenic EPN2 staining is diffuse?
First compare a no-primary control with the stained section to separate detection background from primary-antibody-dependent signal (standard IHC practice). The selected paraffin example used primary antibody at 1:200 overnight at 4°C and secondary antibody at 1:200; treat these as tested image conditions, not a universal optimum (caption A08001-1). If background follows primary incubation, titrate the primary, strengthen protein blocking and washing, and compare adjacent sections under identical retrieval (standard IHC practice). For peroxidase detection, check the peroxidase block and shorten DAB development if diffuse brown signal obscures morphology (standard IHC practice). Preserve any reproducible cytoplasmic puncta during optimization, consistent with EPN2's vesicular location (UniProt O95208).
How should I score EPN2 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and cytoplasmic compartment before scoring, because reported tissue staining includes neuronal cells and EPN2 localizes to cytoplasm and vesicles (HPA tissue IHC; UniProt O95208). For each specimen, record the percentage of positive target cells and staining intensity, then calculate an H-score as the sum of each intensity category multiplied by its percentage (standard IHC practice). If puncta are the endpoint, quantify puncta density per mm² of evaluable target-cell area and apply one segmentation rule across slides (standard image-analysis practice). Normalize counts to the number or area of intact target cells, and exclude folds, edges and necrotic regions before comparing specimens (standard IHC practice).
When should an apparent EPN2-positive section be questioned?
A plausible positive shows cytoplasmic or vesicle-like staining in the assessed cells; EPN2 is cytoplasmic and clathrin-vesicle-associated (UniProt O95208). Compare neuronal staining with the reported medium staining in caudate, cerebral cortex and hippocampus, but do not use that pattern alone as proof of specificity (HPA tissue IHC). Question staining confined to nuclei, tissue edges, necrosis or a no-primary control, and investigate endogenous peroxidase or DAB precipitate before assigning positivity (UniProt O95208; standard IHC practice). HPA rates its tissue antibody evidence Approved yet reports low staining–RNA consistency and presumed off-target binding, so require concordant morphology and an independent specificity check for consequential conclusions (HPA tissue IHC; standard IHC practice).
Boster reagents
Best EPN2 / Epsin-2 IHC Antibodies
A08001-1 has a human paraffin-section IHC image (catalog image caption). IF is listed, with Human, Mouse, and Rat reactivity; no IF image is supplied (catalog applications; catalog reactivity; catalog IF images).
Real IHC data
Anti-Epsin 2 EPN2 Antibody
Cat # A08001-1
A08001-1 will render with an IHC image of paraffin-embedded human small intestinal carcinoma (catalog image caption). It lists IHC and IF applications and Human, Mouse, and Rat reactivity; the payload supplies no IF image (catalog applications; catalog reactivity; catalog IF images).
Which to pick: For tissue IHC, choose A08001-1: its image shows a human paraffin section stained at 1:200 after sodium citrate pH 6.0 retrieval; the fixative is unreported (catalog image caption). For IF, A08001-1 lists a 1:50 dilution, but supplies no IF image or ICC application, and its clone is unreported (catalog IF dilution; catalog IF images; catalog applications; catalog clone). For cross-species work, A08001-1 lists Human, Mouse, and Rat reactivity, while its pictured IHC evidence is human only (catalog reactivity; catalog image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O95208 (EPN2_HUMAN, Epsin-2).
- Human Protein Atlas. EPN2 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. EPN2 subcellular location (ICC-IF): Localized to vesicles..
- Human Protein Atlas. EPN2 antibody validation summary (2 antibodies).
- Novel Biomarkers for Lymph Node Metastasis and Prognosis of Bladder Cancer by Bioinformatics Analysis. ACS omega 2025 — PMC12573180.
- PubMed PMID:10567358 — UniProt-cited evidence.
- PubMed PMID:10470851 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for EPN2 (Epsin-2). Expected localisation Cytoplasmic vesicles expected (UniProt); IHC compartment unreported (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, heat-mediated (datasheet A08001-1). Validated IHC antibody: A08001-1 at 1:100-1:300 (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 small intestinal carcinoma tissue; fixative not specified (datasheet A08001-1); 2. Fixation: Image fixative and duration unreported (datasheet A08001-1); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: Citrate pH 6.0 , 20min (datasheet A08001-1); 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-EPN2, 1:100-1:300 (datasheet A08001-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific). Selected-image fixative and duration unreported (datasheet A08001-1); verify before use.; Caveat: Presumed off-target staining may affect interpretation (HPA tissue IHC); Isoform / epitope: 4 isoforms; no extracellular segment; epitope coverage unknown (UniProt); Regulation: Highest expression in brain (UniProt). Evidence: UniProt O95208, HPA tissue IHC · Approved, HPA ICC-IF · vesicles., Datasheet A08001-1, PMC12573180. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/EPN2/immunohistochemistry-ihc-guide.