This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
ENAH / Protein enabled homolog · IHC design guide
Design Immunohistochemistry for ENAH
Plan ENAH staining in paraffin sections using the IHC-validated antibody. This guide covers tissue patterns, fixation, antigen retrieval, detection and interpretation.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ENAH 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 | Cytoplasm in breast myoepithelial cells; membrane in pancreatic ducts (HPA tissue IHC) | |
| Staining pattern | Breast myoepithelial/prostate basal cytoplasm; pancreatic duct membrane (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A05337-2) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining has medium agreement with RNA expression (HPA tissue IHC) | |
| Regulation | Overexpressed in most primary breast tumors (UniProt) | |
| Isoform / epitope | 3 isoforms; check epitope coverage across variants (UniProt) |
Section 1
Recommended ENAH IHC & IF Protocols
The catalog antibody has an IHC-P protocol (datasheet A05337-2). The published protocols below cover gastric cancer and oral squamous cell carcinoma tissue (PMC5641146; PMC11545229).
| Sample | Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet A05337-2) |
| Fixation | Image fixative and duration unreported (datasheet A05337-2); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A05337-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A05337-2) |
| Primary antibody | Rabbit anti-ENAH, 2-5 μg/ml (datasheet A05337-2) |
| Primary incubation | Overnight at 4 °C (datasheet A05337-2) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A05337-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ENAH-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several tissues including breast myoepithelial cells, basal prostate epithelium and membranous expression in pancreatic acinar duct epithelium. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet A05337-2); the cited excerpts do not specify retrieval conditions (PMC5641146; PMC11545229).
Section 2
What Is the Expected ENAH Staining Pattern?
ENAH is a cytoplasmic, actin-associated protein found at cell projections and focal adhesions; it has no transmembrane segment (UniProt Q8N8S7). In paraffin sections, expect staining in glandular and epithelial cells, including endometrial glands and breast myoepithelial cells (HPA tissue IHC). HPA rates the tissue IHC evidence Enhanced, while reporting medium consistency between staining and RNA expression (HPA tissue IHC).
| Strong cytoplasmic staining in endometrial glandular cells. | This fits an HPA high-staining cell population and ENAH’s cytoplasmic location (HPA tissue IHC; UniProt Q8N8S7). Judge the pattern within the identified glands, since HPA reports staining by cell type rather than treating every cell in a section as equally positive (HPA tissue IHC). |
| A membrane-associated edge or focal pattern accompanies cytoplasmic staining. | This can fit ENAH at cell projections and focal adhesions (UniProt Q8N8S7). HPA also describes membranous staining in pancreatic acinar duct epithelium (HPA tissue IHC). The exact edge pattern can be harder to resolve with chromogenic IHC than with ICC-IF (general IHC/IF practice). |
| Staining is predominantly nuclear, with little cytoplasmic signal. | Treat this as a compartment mismatch requiring verification: the supplied localisation records place ENAH in cytoplasm, projections and adhesions, and report no nuclear location (UniProt Q8N8S7; HPA subcellular). Review morphology and controls before assigning the nuclear signal to ENAH (general IHC practice). |
| The strongest signal appears in an unexpected cell population, or broadly across the section. | Check whether the cells match the reported tissue pattern before scoring them as positive (HPA tissue IHC). Unexpected staining can reflect cross-reactivity or endogenous detection activity; an omitted-primary control can help assess detection-system background (general IHC practice). |
| Endometrial glandular cells show no discernible staining. | These cells are listed as high staining, so a blank result warrants a technical check, though HPA’s Enhanced rating is not a guarantee for every specimen (HPA tissue IHC). Review tissue identity, morphology and the antibody’s IHC-P procedure, then examine positive and omitted-primary controls (general IHC practice). |
💡Expected ENAH appearanceCall a result positive when identified endometrial glandular cells show clear, relatively strong cytoplasmic signal (HPA: High in endometrial glandular cells; UniProt Q8N8S7 localisation); isolated nuclear staining or indiscriminate section-wide colour calls for control review (general IHC practice).
| Cell compartment and topology | ENAH is cytoplasmic and associated with projections and focal adhesions, without a transmembrane segment (UniProt Q8N8S7). Thus, a membrane-associated pattern should be interpreted alongside cell identity and cytoplasmic staining; membrane colour alone does not establish a transmembrane target (UniProt Q8N8S7; general IHC practice). |
| Choice of tissue and cell population | HPA reports high staining in endometrial glandular cells and several other glandular or epithelial populations, but low staining in cardiomyocytes and skeletal myocytes (HPA tissue IHC). Low is not absent: neither low-staining tissue is established here as a true negative control (HPA tissue IHC). |
| Antibody validation | HPA028448 and HPA028696 each have an Enhanced IHC rating (HPA antibodies). That supports use of their reported patterns as a reference, while HPA’s medium staining–RNA consistency warrants checking the actual cell pattern and controls in each run (HPA tissue IHC; general IHC practice). |
| Isoforms and epitope coverage | UniProt lists three ENAH isoforms (UniProt Q8N8S7). The supplied records do not identify this catalog antibody’s epitope or its recognition of each isoform; a missing signal cannot be assigned to isoform choice from these records alone (UniProt Q8N8S7; supplied antibody evidence). |
| Antigen retrieval | Retrieval is a general paraffin-section workflow variable; follow the IHC-validated antibody’s documented conditions and compare controls when adjusting it (general IHC practice). The supplied HPA and UniProt records do not establish an ENAH-specific fixation effect or retrieval requirement (HPA tissue IHC; UniProt Q8N8S7). |
| Q: Does ICC-IF localisation define the IHC protocol? | A: No. ICC-IF places ENAH at plasma membrane, focal adhesion sites and cytosol, providing a compartment check (HPA subcellular). It does not supply paraffin-section conditions; use the catalog antibody’s IHC-P procedure for the IHC workflow (HPA subcellular; general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| Known high-staining glandular cells are blank. | An assay or specimen problem is possible; HPA reports high staining in endometrial glandular cells (HPA tissue IHC). | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. |
| Nuclear signal dominates the slide. | The pattern conflicts with the reported cytoplasmic, projection and adhesion locations (UniProt Q8N8S7; HPA subcellular). | Inspect an omitted-primary control and repeat with the IHC-validated antibody’s documented conditions; score nuclear-only colour cautiously until its source is resolved (general IHC practice). |
| Colour is diffuse across tissue or appears in the omitted-primary control. | Nonspecific detection or endogenous enzyme activity may contribute to chromogenic background (general IHC practice). | Review blocking, washes, detection chemistry and endogenous-activity quenching appropriate to the detection system; compare the control with identified positive cells (general IHC practice; HPA tissue IHC). |
| An unexpected cell type stains more strongly than the expected population. | Cell identification may be wrong, or staining may reflect cross-reactivity or endogenous detection activity (general IHC practice). | Recheck morphology and the tissue-specific HPA cell assignments; compare an omitted-primary control and, if available, staining from an independently validated ENAH antibody (HPA tissue IHC; HPA antibodies; general IHC practice). |
| Low-staining muscle cells show faint colour. | HPA lists cardiomyocytes and skeletal myocytes as low, so faint signal alone does not establish an artefact or a negative-control failure (HPA tissue IHC). | Assess localisation, morphology and control background before scoring; reserve a true negative designation for a control established by the assay (HPA tissue IHC; general IHC practice). |
| IHC seems less sharply peripheral than an ICC-IF image. | ICC-IF resolves plasma membrane and focal adhesion sites, while chromogenic tissue sections may show those details less clearly (HPA subcellular; general IHC/IF practice). | Judge the IHC result against its cell-specific tissue pattern and controls, and use ICC-IF localisation only as supporting context (HPA tissue IHC; HPA subcellular; general IHC practice). |
Experiment evidence
IHC & IF images showing ENAH staining
A05337-2 · IHCIHC analysis of ENAH/MENA using anti-ENAH/MENA antibody (A05337-2). ENAH/MENA was detected in a paraffin-embedded section of human liver can
A05337-2 · IHCIHC analysis of ENAH/MENA using anti-ENAH/MENA antibody (A05337-2). ENAH/MENA was detected in a paraffin-embedded section of human ovarian s
A05337-2 · IFIF analysis of ENAH/MENA using anti-ENAH/MENA antibody (A05337-2). ENAH/MENA was detected in an immunocytochemical section of A431 cells. En
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000154380-ENAH/tissue/cerebral+cortex#imid_7284720
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000154380-ENAH/tissue/colon#imid_7284662
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000154380-ENAH/tissue/liver#imid_7284655
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000154380-ENAH/tissue/kidney#imid_7284656
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA028448; green = ENAH, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA028448; green = ENAH, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ENAH IHC & IF
🧪Run endometrium first; glandular cells should stain (HPA: High in endometrial glandular cells). HPA detects ENAH in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and expect cells outside the stained glandular compartment to show only background in those controls rather than treating them as confirmed ENAH-negative cells (HPA: no negative tissue rows).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: ENAH is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ENAH in A-431, U-251MG, U2OS, hTERT-RPE1 (serum starved), with annotated localisation: Plasma membrane (supported), Focal adhesion sites (enhanced), Cytosol (enhanced) (HPA subcellular).
Technical controls: Include a secondary-only slide and a concentration-matched rabbit immunoglobulin control (caption: rabbit primary antibody), plus an ENAH-knockout sample or a validated immunizing-peptide block as a biological specificity control (standard IHC practice). Check endogenous peroxidase and biotin background in the endometrial slide (caption: SABC/DAB detection).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A05337-2 paraffin-section caption does not state the fixative (caption: fixative unreported). The caption uses heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required; it gives no basis to judge whether frozen sections or IF would be easier (caption: paraffin-section IHC). In endometrial glandular cells, endogenous peroxidase or biotin could complicate interpretation of DAB staining, so assess that background with the detection controls (HPA: High in endometrial glandular cells; caption: SABC/DAB detection).
HPA tissue IHC evidence for ENAH
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data. 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 | High | Protein (IHC) | HPA → |
| Cerebellum | Bergmann glia - cytoplasm/membrane | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Endometrium | Glandular cells | High | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: ENAH is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced ENAH IHC Tips
Troubleshoot ENAH chromogenic IHC in paraffin sections by checking retrieval, cell type, subcellular pattern and scoring before interpreting staining.
What retrieval conditions should I start with for weak ENAH staining in paraffin sections?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 (datasheet A05337-2). The selected paraffin-section example used that retrieval before overnight incubation at 4°C with 2 μg/ml antibody (datasheet A05337-2). Keep retrieval conditions consistent across a comparison set, and include a reference section in each run so a change in staining can be distinguished from a change in tissue. If staining remains weak, adjust heating duration in a small pilot while checking tissue morphology; record the duration and cooling conditions for reproducibility (standard IHC practice).
How should I investigate weak ENAH staining that varies between paraffin blocks?
The selected paraffin-section caption does not report its fixative, and target-specific ENAH sensitivity to fixation is unknown (datasheet A05337-2). Compare blocks with documented processing histories using the same EDTA pH 8.0 retrieval, antibody concentration and detection run (datasheet A05337-2; standard IHC practice). Include a reference section on each slide batch, then assess staining alongside preserved tissue morphology and cell-type identification (standard IHC practice). If processing histories differ, treat a block-to-block signal difference as unresolved until matched sections or another validation method support a biological difference; tissue staining patterns alone cannot establish a fixation effect (standard IHC practice).
Which subcellular staining patterns are plausible for ENAH in tissue sections?
Assess ENAH chiefly as cytoplasmic staining, with possible membrane-associated or junctional enrichment where cellular detail permits (UniProt Q8N8S7 subcellular location; HPA tissue IHC profile). ENAH is associated with the cytoskeleton, lamellipodia, filopodia and focal adhesions, while the HPA subcellular profile reports plasma membrane, cytosol and focal adhesion sites (UniProt Q8N8S7 subcellular location; HPA subcellular). In paraffin sections, avoid requiring a sharply resolved leading edge, because section orientation and chromogenic resolution can obscure fine projections (standard IHC practice). Review localisation within identified cells and compare reference tissue on the same run; isolated nuclear staining needs separate validation (standard IHC practice; UniProt Q8N8S7 subcellular location).
Can this stain distinguish ENAH isoforms or phosphorylation states?
Treat the chromogenic signal as ENAH immunoreactivity unless the antibody epitope and its isoform coverage have been established separately (standard IHC practice). The record lists 3 ENAH isoforms and a WH1 domain at residues 1–111 (UniProt Q8N8S7 isoforms and domains). It also lists modified residues, including phosphorylation at 125, 265, 502, 506 and 508, but those annotations do not demonstrate that this antibody distinguishes modification states (UniProt Q8N8S7 modified residues; standard IHC practice). For an isoform or phospho-specific claim, obtain the epitope sequence and validate specificity with appropriate controls before comparing stained sections (standard IHC practice).
How should I adapt the ENAH tissue question for multiplex immunofluorescence?
Use IF as a separate validation workflow: the selected antibody example establishes chromogenic staining in a paraffin section, with no IF conditions supplied (datasheet A05337-2). In breast tissue, pair ENAH with an independently validated myoepithelial marker to identify the expected cells during multiplex analysis (HPA tissue IHC profile; standard IF practice). Select a fluorophore channel after inspecting unstained tissue autofluorescence, and include single-label controls to assess bleed-through (standard IF practice). Because ENAH has no transmembrane segment and is reported in cytosol and focal adhesions, test mild permeabilisation when the antibody recognises an intracellular epitope; its exact epitope is unspecified here (UniProt Q8N8S7 topology and subcellular location; standard IF practice).
What should I check when ENAH staining is diffuse or the DAB background is high?
First compare the stained section with a no-primary control and inspect whether background follows tissue edges, damaged regions or a particular cell population (standard IHC practice). The selected example uses a 10% goat serum block, biotinylated secondary antibody, streptavidin–biotin detection and DAB (datasheet A05337-2). For that workflow, check peroxidase blocking and consider endogenous biotin as potential sources of signal independent of ENAH (standard IHC practice). Titrate antibody or shorten chromogen development in a controlled pilot while retaining a reference section; interpret any remaining cytoplasmic or membrane-associated pattern against expected cell types (standard IHC practice; HPA tissue IHC profile).
How can I score ENAH IHC across samples without conflating cell composition and intensity? ⚠ ANSWER MARKED FOR VERIFICATION
Define the compartment and cell population before scoring, then keep those definitions fixed across sections (standard IHC practice). For epithelial or myoepithelial populations, report the percentage of positive cells and an H-score based on intensity categories of 0–3; record cytoplasmic and membrane-associated staining separately when both are visible (standard IHC practice; HPA tissue IHC profile). If counting discrete positive cells, report density per mm² of viable, annotated tissue and exclude folds or necrosis (standard IHC practice). Normalise comparisons to the same cell type and sampled viable area, and include a reference section across staining runs (standard IHC practice).
How do I decide whether an apparent ENAH-positive area is biological signal or artefact?
A plausible result places staining in identifiable cells with cytoplasmic or membrane-associated distribution, consistent with reported ENAH localisation (UniProt Q8N8S7 subcellular location; HPA tissue IHC profile). Cell identity matters: HPA reports cytoplasmic expression in breast myoepithelial and basal prostate epithelial cells, and membranous expression in pancreatic acinar duct epithelium (HPA tissue IHC profile). Question isolated nuclear signal, staining confined to section edges or necrosis, and deposits repeated in the no-primary control (standard IHC practice; UniProt Q8N8S7 subcellular location). Check peroxidase-dependent background before calling DAB signal positive, and seek independent validation for unexpected cells or compartments (standard IHC practice).
Boster reagents
Best ENAH / Protein enabled homolog IHC Antibodies
A05337-2 has human paraffin-section IHC images and an A431-cell IF image (catalog image captions); listed reactivity spans Human, Mouse, and Rat (catalog reactivity).
Real IHC data
Anti-ENAH/MENA Antibody ®
Cat # A05337-2
A05337-2 has IHC images from paraffin-embedded human liver cancer, ovarian serous adenocarcinoma, and renal pelvis squamous metaplasia tissue (catalog IHC captions). A05337-2 has an IF image in A431 cells and lists both IF and ICC applications (catalog IF caption; catalog applications).
Which to pick: For tissue IHC, choose A05337-2: its human paraffin-section captions document EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody; the fixative is unreported (catalog IHC captions). For IF/ICC, A05337-2 lists both applications and has an A431-cell IF image (catalog applications; catalog IF caption). For cross-species work, A05337-2 lists Human, Mouse, and Rat reactivity, but the supplied IHC images show human tissue only; clone information is unreported (catalog reactivity; catalog IHC captions; catalog clone field).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8N8S7 (ENAH_HUMAN, Protein enabled homolog).
- Human Protein Atlas. ENAH tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. ENAH subcellular location (ICC-IF): Localized to the plasma membrane, cytosol and focal adhesion sites..
- Human Protein Atlas. ENAH antibody validation summary (2 antibodies).
- The significance of ENAH in carcinogenesis and prognosis in gastric cancer. Oncotarget 2017 — PMC5641146.
- Enah overexpression is correlated with poor survival and aggressive phenotype in gastric cancer. Cell death & disease 2018 — PMC6155292.
- Upregulation of ENAH by a PI3K/AKT/β-catenin cascade promotes oral cancer cell migration and growth via an ITGB5/Src axis. Cellular & molecular biology letters 2024 — PMC11545229.
- Circ_0003340 regulates the expression of ENAH to affect the development of esophageal cancer through miR-874-3p. Thoracic cancer 2023 — PMC10040281.
- PubMed PMID:15027125 — UniProt-cited evidence.
- PubMed PMID:23129656 — UniProt-cited evidence.
- PubMed PMID:16710414 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ENAH (Protein enabled homolog). Expected localisation Cytoplasm in breast myoepithelial cells; membrane in pancreatic ducts (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A05337-2). Validated IHC antibody: A05337-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet A05337-2); 2. Fixation: Image fixative and duration unreported (datasheet A05337-2); 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: EDTA pH 8.0 (datasheet A05337-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A05337-2); 8. Primary antibody: Rabbit anti-ENAH, 2-5 μg/ml (datasheet A05337-2); 9. Primary incubation: Overnight at 4 °C (datasheet A05337-2). Watch for: Fixation: Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining has medium agreement with RNA expression (HPA tissue IHC); Isoform / epitope: 3 isoforms; check epitope coverage across variants (UniProt); Regulation: Overexpressed in most primary breast tumors (UniProt). Evidence: UniProt Q8N8S7, HPA tissue IHC · Enhanced, HPA ICC-IF · plasma membrane, cytosol and focal adhesion sites., Datasheet A05337-2, PMC5641146, PMC11545229. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ENAH/immunohistochemistry-ihc-guide.