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ENO2 / Gamma-enolase · IHC design guide
Design Immunohistochemistry for ENO2
Plan ENO2 IHC in paraffin sections using the cytoplasmic staining reported in CNS, peripheral nerves and pancreatic islets (HPA tissue IHC). This guide covers fixation, controls and scoring by cell type and compartment.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ENO2 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 tissue staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in CNS, nerves and islets (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A02930) | |
| 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 | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A02930) | |
| Caveat | Pancreatic endocrine cells also stain (HPA tissue IHC) | |
| Regulation | Dimers vary by cell type and development (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage is unknown (UniProt) |
Section 1
Recommended ENO2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet A02930) is accompanied by two published ENO2 IHC protocols (PMC8253290; PMC9367517).
| Sample | Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet A02930) |
| Fixation | Image fixative and duration unreported (datasheet A02930); 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, 20 min (datasheet A02930) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A02930) |
| Primary antibody | Rabbit anti-ENO2, 0.5-1μg/ml (datasheet A02930) |
| Primary incubation | Overnight at 4 °C (datasheet A02930) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A02930) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ENO2-positive staining in neuronal cells of caudate (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in CNS, peripheral nerves and pancreatic islets. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6 heat retrieval (datasheet A02930); the published high-pressure citrate step offers a timed starting point (PMC9367517).
Section 2
What Is the Expected ENO2 Staining Pattern?
ENO2 should stain mainly the cytoplasm of CNS neuronal cells, cerebellar granular-layer cells and pancreatic endocrine cells; peripheral nerves may show lower expression (HPA tissue IHC). The HPA tissue profile has Enhanced reliability, reflecting consistency between staining and RNA data (HPA tissue IHC). Membrane-associated signal is possible because ENO2 can translocate to the plasma membrane, although it has no transmembrane segment (UniProt P09104 topology and subcellular location).
| Strong cytoplasmic staining in caudate, cerebral-cortex or hippocampal neuronal cells. | This matches the reported high staining in those cell populations (HPA tissue IHC). Compare cells within the section as well as the overall tissue signal; the expected pattern is cellular and predominantly cytoplasmic (HPA tissue IHC). |
| Predominantly nuclear staining, with little cytoplasmic signal in expected positive cells. | A nuclear-dominant pattern does not match the reported cytoplasmic tissue profile (HPA tissue IHC). Review the negative control, detection background and whether the signal follows cell boundaries before interpreting it as ENO2 (standard IHC practice). |
| Strong staining in a cell population reported as undetected, such as adipocytes in adipose tissue. | That disagrees with the specified HPA cell-level observation (HPA tissue IHC: adipocytes not detected). Consider antibody cross-reactivity or endogenous chromogenic detection activity; a negative call for one cell population does not classify every cell in that tissue (standard IHC practice). |
| Diffuse color across tissue and blank areas, obscuring cell boundaries. | This is background rather than a readable cellular distribution (standard IHC practice). Check a no-primary control and review blocking, washes and detection conditions before assigning ENO2-positive cells; HPA's tissue profile describes cytoplasmic expression in defined cell populations (HPA tissue IHC). |
| No staining in neuronal cells of a well-preserved caudate, cortex or hippocampus section. | Those neuronal cells are reported as High by HPA (HPA tissue IHC). First suspect a failed staining run or unsuitable assay conditions; absence on one slide cannot establish biological loss of ENO2 without a working positive control (standard IHC practice). |
💡Expected ENO2 appearanceCall a section positive when expected neuronal cells show strong, predominantly cytoplasmic signal (HPA tissue IHC); isolated nuclear-dominant or diffuse color is a suspect pattern requiring controls (standard IHC practice).
| Cell population and tissue | HPA reports High neuronal staining in caudate, cerebral cortex and hippocampus, and granular-layer cell staining in cerebellum; pancreatic endocrine cells are Medium (HPA tissue IHC). Interpret intensity against the relevant cell population. |
| Subcellular distribution | The tissue IHC profile is cytoplasmic (HPA tissue IHC). UniProt also lists cytoplasm and cell membrane and describes plasma-membrane translocation; ENO2 has no transmembrane segment (UniProt P09104). Membrane signal alone should not replace the expected cytoplasmic tissue pattern. |
| Antibody validation | HPA lists IHC status as Enhanced for CAB000063 and CAB073539, and Supported for CAB079023 (HPA antibodies). These statuses support pattern assessment for those antibodies; they do not specify conditions or establish that every antibody produces the same stain. |
| Enolase subunit context | Mammalian enolase has alpha, beta and gamma subunits in cell-type-dependent dimers; gamma-containing dimers occur in neurons (UniProt P09104 tissue specificity and subunit). Interpret unexpected staining with antibody specificity and tissue controls in mind. |
| Isoforms and processing | UniProt lists 2 ENO2 isoforms, no signal peptide, no propeptide and a gamma-enolase chain spanning residues 2–434 (UniProt P09104). The payload gives no antibody epitope, so it does not establish isoform coverage or a processing-dependent IHC pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected neuronal positive control has no signal. | The result conflicts with HPA's High neuronal staining in caudate, cortex and hippocampus (HPA tissue IHC); a staining-run problem remains possible (standard IHC practice). | Confirm tissue identity and preservation, then check primary-antibody application, detection reagents and counterstain against a known working run (standard IHC practice). |
| Color is diffuse or appears in a no-primary control. | Background may arise from detection chemistry or insufficient blocking and washing (standard IHC practice). | Review endogenous-enzyme blocking where relevant, washes and detection time; repeat with a no-primary control before scoring cellular signal (standard IHC practice). |
| A nuclear-dominant pattern replaces cytoplasmic staining. | That compartment conflicts with the cytoplasmic tissue profile (HPA tissue IHC); nonspecific staining or an interpretation error is possible (standard IHC practice). | Compare the positive and no-primary controls, examine cell boundaries, and confirm the antibody's IHC validation before assigning the signal to ENO2 (HPA antibodies; standard IHC practice). |
| Adipocytes in adipose tissue stain strongly. | HPA reports ENO2 as not detected in that specific cell population (HPA tissue IHC); cross-reactivity or endogenous detection activity may explain the color (standard IHC practice). | Check a no-primary control and compare a known positive neuronal section in the same run; interpret the adipocyte result at cell level (HPA tissue IHC; standard IHC practice). |
| Expected cells stain weakly or unevenly. | The payload provides no ENO2-specific fixation sensitivity or retrieval setting; uneven staining can reflect general section or assay variation (standard IHC practice). | Compare sections with a positive control and review section quality, retrieval consistency and detection timing using the validated IHC workflow (standard IHC practice). |
| Q: Can the IF/ICC localisation be used to judge this IHC slide? | HPA reports plasma membrane and cytosol in ICC-IF but cautions that its summary uses antibodies targeting proteins from multiple genes (HPA subcellular). | A: Use the cytoplasmic tissue IHC profile as the primary slide comparator; treat the ICC-IF localisation as context and consult the separate IF/ICC guide for that application (HPA tissue IHC; HPA subcellular). |
Experiment evidence
IHC & IF images showing ENO2 staining
A02930 · IHCIHC analysis of NSE using anti-NSE antibody (A02930). NSE was detected in paraffin-embedded section of human lung cancer tissue. Heat mediat
A02930 · IHCIHC analysis of NSE using anti-NSE antibody (A02930). NSE was detected in paraffin-embedded section of human placenta tissue. Heat mediated
A02930 · IFIF analysis of NSE using anti-NSE antibody (A02930). NSE was detected in immunocytochemical section of A431 cells. Enzyme antigen retrieval
PA1061 · IHCIHC analysis of NSE using anti-NSE antibody (PA1061). NSE was detected in paraffin-embedded section of rat brain tissue. Heat mediated antig
PA1061 · IHCIHC analysis of NSE/ENO2 using anti-NSE/ENO2 antibody (PA1061). NSE/ENO2 was detected in a paraffin-embedded section of human brain tissue.
M02930-1 · IHCImmunohistochemical analysis of paraffin-embedded human uterus cancer, using NSE Antibody.
M02930-1 · IFImmunofluorescent analysis of A431 cells, using NSE Antibody.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000111674-ENO2/tissue/cerebral+cortex#imid_255090
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000111674-ENO2/tissue/colon#imid_202688
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000111674-ENO2/tissue/liver#imid_202681
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000111674-ENO2/tissue/kidney#imid_202674
HPA · ICC-IF HeLaHuman Protein Atlas immunofluorescence, HeLa, antibody HPA068284; green = ENO2, blue = nucleus, red = microtubules.
HPA · ICC-IF HeLaHuman Protein Atlas immunofluorescence, HeLa, antibody HPA068284; green = ENO2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ENO2 IHC & IF
🧪Run caudate first; neuronal cells should stain strongly (HPA: High in caudate neuronal cells). Use adipose tissue adipocytes as the negative comparator (HPA: Not detected in adipocytes); on the caudate slide, assess non-neuronal cells for background-level staining, while recognizing that HPA does not establish them as ENO2-negative (HPA: caudate neuronal-cell row).
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 ENO2 in HeLa, U2OS, A-431, PC-3, with annotated localisation: Plasma membrane (enhanced), Cytosol (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, an isotype control matched to the primary antibody’s host and clonality, and an ENO2 knockout control or peptide-block control if the immunizing peptide is available (standard IHC practice; caption: rabbit primary antibody). Block endogenous peroxidase and check for endogenous biotin before interpreting chromogenic signal (caption: SABC/DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A02930 tissue-IHC caption does not state a fixative (caption: fixative not stated). The reported paraffin-section procedure uses citrate retrieval at pH 6 for 20 minutes, but the caption does not establish that retrieval is required under other conditions (caption: citrate retrieval). Whether frozen sections or IF are easier is unreported; for caudate, assess background in non-neuronal cells and endogenous peroxidase or biotin signal when scoring neuronal staining (HPA: High in caudate neuronal cells; caption: SABC/DAB detection; standard IHC practice).
HPA tissue IHC evidence for ENO2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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 granular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Hippocampus | Neuronal cells | High | Protein (IHC) | HPA → |
| Pancreas | Pancreatic endocrine 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 | Endocrine 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 ENO2 IHC Tips
Troubleshoot ENO2 staining in paraffin sections by checking retrieval, cellular pattern and controls against the reported tissue distribution.
How should I retrieve ENO2 in paraffin sections with weak staining?
Start with heat-mediated retrieval in citrate buffer at pH 6 for 20 minutes (datasheet A02930). This is the retrieval used for the catalog antibody in a paraffin section of human lung cancer; the caption does not state the fixative (datasheet A02930). If staining remains weak, compare a modestly longer heating time on matched sections while keeping the buffer and detection conditions constant (standard IHC practice). Include a known positive neuronal section so that weak staining in the study tissue is distinguishable from a failed run (HPA: high ENO2 in caudate neuronal cells; standard IHC practice). Excessive heating can damage tissue morphology and complicate cellular scoring (standard IHC practice).
Could fixation explain weak ENO2 staining in my paraffin sections?
Target-specific fixation sensitivity for ENO2 is unknown from the supplied evidence; the catalog image describes a paraffin section but does not report its fixative (datasheet A02930). Record the fixative, fixation duration and section processing for each batch before comparing staining intensity (standard IHC practice). If weak staining tracks with a processing batch, stain matched positive-control sections alongside study sections using the same citrate pH 6 retrieval and antibody conditions (datasheet A02930; standard IHC practice). Compare morphology and control staining before changing retrieval time, since damaged sections can make an apparent loss of cytoplasmic signal hard to interpret (HPA: cytoplasmic tissue expression; standard IHC practice). Do not assign a fixation mechanism from the tissue expression pattern alone (standard IHC practice).
Is membrane staining compatible with ENO2 expression in chromogenic IHC?
A predominantly cytoplasmic pattern in neuronal cells and pancreatic endocrine cells fits the reported tissue IHC profile (HPA: tissue IHC profile). Membrane-associated ENO2 is biologically plausible because the protein can translocate to the plasma membrane, although it has no annotated transmembrane segment (UniProt P09104: subcellular location and topology). Evaluate membrane accentuation together with cytoplasmic staining, cell identity and matched controls before calling it specific (standard IHC practice). Subcellular IF evidence lists plasma membrane and cytosol, but its antibody evidence carries a multiple-gene targeting caution (HPA: subcellular summary). Diffuse chromogen outside intact cells or along torn section edges should prompt a check of background and morphology (standard IHC practice).
Can this stain distinguish ENO2 isoforms or dimer partners?
Do not score the chromogenic signal as a particular ENO2 splice isoform: 2 isoforms are listed, but the supplied antibody evidence does not map its epitope to either one (UniProt P09104: isoforms; datasheet A02930). Enolase gamma subunits can occur in alpha/gamma and gamma/gamma dimers in neurons, and dimer composition alone cannot be read from a stained section (UniProt P09104: tissue specificity and subunit; standard IHC interpretation). ENO2 has annotated modified residues, including phosphorylation and acetylation, but their effect on this antibody's binding is unreported (UniProt P09104: modified residues; datasheet A02930). Resolve an isoform-specific claim only with independently documented epitope specificity and a suitable orthogonal assay (standard IHC practice).
How should I follow up an ENO2 IHC pattern with multiplex IF?
Use multiplex IF as a separate follow-up to the chromogenic section: pair ENO2 with a marker for the expected neuronal or pancreatic endocrine cell population and check single-marker controls (HPA: tissue IHC profile; standard IF practice). Choose fluorophores whose signal can be separated from the tissue's measured autofluorescence, particularly when comparing weak cytosolic and membrane-associated signal (UniProt P09104: subcellular location; standard IF practice). For an intracellular epitope, test gentle permeabilisation; for an extracellularly accessible membrane epitope, compare staining without permeabilisation, but the antibody's epitope side is not supplied (standard IF practice; datasheet A02930). The reported subcellular IF pattern includes cytosol and plasma membrane, with a multiple-gene antibody caution (HPA: subcellular summary).
What should I check when ENO2 DAB staining is widespread?
First compare the distribution with a no-primary control and inspect whether chromogen follows cells, section edges or damaged tissue (standard IHC practice). The catalog image used a 10% goat serum block, 1 µg/ml primary antibody overnight at 4°C, a biotinylated secondary and DAB detection (datasheet A02930). For a peroxidase-based DAB workflow, check the peroxidase block and secondary-only background; these are general detection controls, not ENO2-specific findings (standard IHC practice). If staining remains diffuse, titrate the primary and review wash conditions while retaining a positive-control section (standard IHC practice). Widespread signal in cells expected to lack detectable ENO2, such as adipocytes, warrants closer scrutiny (HPA: adipocytes not detected; standard IHC interpretation).
How can I quantify ENO2 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, because ENO2 expression differs between neuronal cells, pancreatic endocrine cells and some other sampled populations (HPA: tissue IHC profile). For cellular staining, report the percentage of positive target cells and an H-score based on intensity categories; for sparse positive cells, density per mm² of viable tissue can be useful (standard IHC practice). Normalise cell counts to the number of evaluable target cells or measured viable area, and apply the same thresholds across batches (standard IHC practice). Keep cytoplasmic and membrane scores separate if both patterns are being tested, given ENO2's reported locations (UniProt P09104: subcellular location; standard IHC practice). Exclude folds, edges and necrotic regions using prespecified criteria (standard IHC practice).
How do I separate true ENO2 positivity from staining artefact?
Give greatest weight to cellular cytoplasmic staining in the expected population, such as caudate neuronal cells, when the positive control also works (HPA: high in caudate neuronal cells; standard IHC practice). Membrane staining can be compatible with ENO2, but isolated outlines without convincing cellular context need corroboration (UniProt P09104: subcellular location; standard IHC interpretation). Check unexpected staining in a comparator population reported as undetected, such as bronchial respiratory epithelial cells, against morphology and controls rather than treating the atlas pattern as an absolute rule (HPA: bronchus not detected; standard IHC practice). Edge deposits, necrotic areas and signal in no-primary or secondary-only controls favour artefact; residual endogenous peroxidase can also produce DAB signal (standard IHC practice).
Boster reagents
Best ENO2 / Gamma-enolase IHC Antibodies
Catalog anti-ENO2 antibodies have paraffin-section IHC images from human lung cancer, placenta, brain, and uterus cancer, rat and mouse brain, plus A431 IF images (catalog image captions).
Real IHC data
Anti-NSE/ENO2 Antibody ®
Cat # A02930
Real IHC data
Anti-NSE/ENO2 Antibody ®
Cat # PA1061
Real IHC data
Anti-NSE ENO2 Rabbit Monoclonal Antibody
Cat # M02930-1
A02930 has IHC images from human lung cancer and placenta and rat and mouse brain, plus an A431 IF image; PA1061 has rat and human brain IHC images (catalog image captions). M02930-1 has a human uterus cancer IHC image and an A431 IF image (catalog image captions).
Which to pick: For tissue IHC, A02930 has the broadest set of pictured paraffin sections; PA1061 offers rat and human brain examples (A02930 and PA1061 IHC captions). For IF/ICC, A02930 has an A431 IF image and a 5 μg/ml recommendation; M02930-1 is a rabbit monoclonal alternative with an A431 IF image (A02930 IF caption and datasheet; M02930-1 catalog and IF caption). All three list human, mouse, and rat reactivity for cross-species selection; their IHC captions show paraffin sections but do not report the fixative (catalog reactivity; IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P09104 (ENOG_HUMAN, Gamma-enolase).
- Human Protein Atlas. ENO2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. ENO2 subcellular location (ICC-IF): Localized to the plasma membrane and cytosol. Caution: Based on antibodies targeting proteins from multiple genes..
- Human Protein Atlas. ENO2 antibody validation summary (5 antibodies).
- Comprehensive Analysis of the Relationship Between Metabolic Reprogramming and Immune Function in Prostate Cancer. OncoTargets and therapy 2021 — PMC8140915.
- Specific activation of glycolytic enzyme enolase 2 in BRAF V600E-mutated colorectal cancer. Cancer science 2021 — PMC8253290.
- ENO2 Promotes Colorectal Cancer Metastasis by Interacting with the LncRNA CYTOR and Activating YAP1-Induced EMT. Cells 2022 — PMC9367517.
- Increased neuron specific enolase expression by urothelial cells exposed to or malignantly transformed by exposure to Cd²⁺ or As³⁺. Toxicology letters 2012 — PMC3390755.
- PubMed PMID:3208766 — UniProt-cited evidence.
- PubMed PMID:3385803 — UniProt-cited evidence.
- PubMed PMID:2792767 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ENO2 (Gamma-enolase). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A02930). Validated IHC antibody: A02930 at 0.5-1μg/ml (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 lung cancer tissue; fixative not specified (datasheet A02930); 2. Fixation: Image fixative and duration unreported (datasheet A02930); 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 , 20 min (datasheet A02930); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A02930); 8. Primary antibody: Rabbit anti-ENO2, 0.5-1μg/ml (datasheet A02930); 9. Primary incubation: Overnight at 4 °C (datasheet A02930). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A02930); Caveat: Pancreatic endocrine cells also stain (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage is unknown (UniProt); Regulation: Dimers vary by cell type and development (UniProt). Evidence: UniProt P09104, HPA tissue IHC · Enhanced, HPA ICC-IF · plasma membrane and cytosol. Caution: Based on antibodies targeting…, Datasheet A02930, PMC8253290, PMC9367517. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ENO2/immunohistochemistry-ihc-guide.