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ENO2 / Gamma-enolase · IHC design guide
Design Immunohistochemistry for ENO2
Plan ENO2 chromogenic IHC in paraffin sections using cytoplasmic CNS staining as a reference (HPA tissue IHC). Compare neuronal and pancreatic endocrine staining with the catalog antibody’s validated IHC conditions (HPA tissue IHC; datasheet A02930).
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); membrane translocation is possible (UniProt) | |
| Staining pattern | Cytoplasmic staining in CNS, peripheral 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 | Keep fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Pancreatic endocrine cells stain less strongly than CNS neurons (HPA tissue IHC) | |
| Regulation | Enolase dimers vary with cell type and development (UniProt) | |
| Isoform / epitope | 2 isoforms; check epitope coverage across variants (UniProt) |
Section 1
Recommended ENO2 IHC & IF Protocols
The catalog antibody protocol and four published ENO2 IHC protocols provide starting conditions for paraffin sections (datasheet A02930; PMC8140915; PMC8253290; PMC9367517; PMC3390755).
| 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 heat-mediated citrate retrieval at pH 6 (datasheet A02930); three published ENO2 IHC methods also use citrate retrieval (PMC8140915; PMC9367517; PMC3390755).
Section 2
What Is the Expected ENO2 Staining Pattern?
In paraffin-section IHC, expect ENO2 mainly in the cytoplasm of CNS neurons, cells in the cerebellar granular layer, peripheral nerves, and pancreatic islets (HPA tissue IHC: Enhanced; cytoplasmic profile). Neuronal staining is high in the sampled CNS regions, while pancreatic endocrine-cell staining is medium (HPA tissue IHC). ENO2 can also reach the cell membrane despite having no transmembrane segment (UniProt P09104: location and topology).
| Strong cytoplasmic staining in cortical, hippocampal, or caudate neurons. | This matches the High neuronal staining reported in these regions (HPA tissue IHC). Judge the result by its cellular distribution as well as intensity: the expected signal occupies neuronal cytoplasm (HPA tissue IHC). A membrane edge can be plausible because ENO2 can translocate to the plasma membrane (UniProt P09104: location). |
| Predominantly nuclear staining, with little neuronal cytoplasmic signal. | A nuclear-only pattern does not match the reported cytoplasmic tissue profile (HPA tissue IHC) or the cytoplasm and cell-membrane locations (UniProt P09104). Treat it as a possible artefact and review the controls and staining distribution before scoring it as ENO2. Do not reject a membrane-associated rim solely for being outside the cytoplasm (UniProt P09104: location). |
| Strong staining in adipocytes or respiratory epithelial cells. | Those cell types are reported as Not detected in adipose tissue and bronchus, respectively (HPA tissue IHC). If staining persists in them, consider antibody cross-reactivity or, with an enzyme-based detection system, endogenous detection activity (general IHC practice). Compare the suspect cells with a known-positive neuronal region and the assay controls before assigning target-specific signal. |
| Widespread pale color obscures cell boundaries across the section. | Diffuse, poorly localized color is difficult to reconcile with the reported cytoplasmic pattern in CNS, peripheral nerves, and pancreatic islets (HPA tissue IHC). It can reflect nonspecific binding, residual detection activity, or excessive chromogen development (general IHC practice). Interpretation should wait until the background is low enough to distinguish positive cells from surrounding tissue. |
| No signal in a known-positive neuronal region. | Absent staining conflicts with the High neuronal signal reported in caudate, cerebral cortex, and hippocampus (HPA tissue IHC). It does not establish that the specimen lacks ENO2. Check that tissue morphology is preserved, the antibody and detection reagents worked, and the assay conditions follow an IHC-validated protocol (general IHC practice). |
💡Expected ENO2 appearanceCall the stain consistent with ENO2 when neuronal cytoplasm is strongly positive in a known-positive CNS region, with medium cytoplasmic staining possible in pancreatic endocrine cells (HPA tissue IHC); prominent nuclear-only or diffuse tissue-wide color is suspect against that profile (HPA tissue IHC).
| Cell compartment | Cytoplasm is the principal tissue-IHC pattern (HPA tissue IHC). Membrane-associated signal is biologically plausible because ENO2 can translocate there despite lacking a transmembrane segment (UniProt P09104: location and topology). |
| Cell and tissue context | CNS neuronal cells and cerebellar granular-layer cells are High; pancreatic endocrine cells are Medium (HPA tissue IHC). Compare like cell types when judging intensity. |
| Antibody evidence | The tissue-IHC profile is rated Enhanced for consistency with RNA data (HPA tissue IHC). Individual IHC antibody statuses differ: CAB000063 and CAB073539 are Enhanced; CAB079023 is Supported (HPA antibodies). |
| What should IF/ICC show? | HPA reports cytosol and plasma-membrane localization in ICC-IF, but cautions that its summary uses antibodies targeting proteins from multiple genes (HPA subcellular). Use the separate IF/ICC guide for assay design. |
| Situation | Likely cause | Next action |
|---|---|---|
| Neuronal positive control is blank. | A failed staining or detection step is possible; high neuronal staining is expected in sampled CNS regions (HPA tissue IHC). | Confirm tissue identity and morphology, then check reagent activity, the IHC-validated antibody conditions, and detection controls (general IHC practice). |
| Only nuclei appear positive. | The compartment conflicts with the cytoplasmic tissue profile (HPA tissue IHC) and UniProt locations (UniProt P09104). | Check the counterstain and omission control, then reassess whether any specific neuronal cytoplasmic signal remains (general IHC practice). |
| Adipocytes or bronchial epithelium stain strongly. | These cells are reported as Not detected (HPA tissue IHC); cross-reactivity or endogenous detection activity is possible. | Compare with a neuronal positive control; use primary-antibody omission and detection-system controls to investigate the unwanted color (general IHC practice). |
| Color covers cells and extracellular areas uniformly. | Nonspecific binding or overdevelopment may obscure the expected cell-associated pattern (general IHC practice; HPA tissue IHC: cytoplasmic profile). | Review blocking, washes, antibody concentration, and chromogen development; retain settings that resolve individual positive cells (general IHC practice). |
| Pancreatic endocrine cells stain less strongly than CNS neurons. | That difference agrees with Medium endocrine-cell versus High neuronal staining (HPA tissue IHC). | Assess each compartment against its reported level and cell type; do not require pancreatic cells to match neuronal intensity (HPA tissue IHC). |
| A narrow membrane rim accompanies neuronal cytoplasmic staining. | ENO2 can move to the plasma membrane (UniProt P09104: location); HPA ICC-IF also reports membrane and cytosol, with a multi-gene-antibody caution (HPA subcellular). | Score the tissue-IHC cytoplasmic pattern first (HPA tissue IHC), and use controls before interpreting the rim as target-specific (general IHC practice). |
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 as the negative tissue, focusing on adipocytes (HPA: Not detected in adipocytes); on the caudate slide, use morphologically non-neuronal cells as internal background comparators and check for little or no specific staining rather than assuming they are ENO2-negative.
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 no-primary (secondary-only) and host-species-matched isotype controls, matching the primary antibody’s clonality once confirmed (caption: rabbit primary), plus ENO2-knockout material as a biological negative. Quench endogenous peroxidase and check for endogenous biotin with the caption’s biotin-based DAB detection; assess tissue autofluorescence if using IF (caption: biotinylated secondary and SABC–DAB).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A02930 paraffin-section caption does not state a fixative (caption: fixative unreported). Heat retrieval in citrate buffer at pH 6 for 20 minutes was used in that IHC example, but retrieval dependence is unreported (caption: citrate retrieval); the supplied evidence does not establish whether frozen sections or IF are easier. For IF, check caudate tissue autofluorescence before interpreting signal; the reported plasma membrane and cytosol pattern carries a cross-antibody caution (HPA: subcellular summary).
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, compartment, cell identity and matched controls (datasheet A02930; HPA tissue IHC; UniProt P09104).
What should I change when ENO2 staining is weak after antigen retrieval?
Start with heat-mediated retrieval in citrate buffer at pH 6, as specified for the catalog antibody (datasheet A02930). The selected paraffin-section example used 20 minutes of retrieval before overnight incubation with 1 µg/ml primary antibody at 4°C (caption A02930). If staining remains weak, compare a modestly longer retrieval with the original condition on matched sections, while checking tissue preservation and a positive neuronal control (standard IHC practice; HPA tissue IHC: high in cortical neuronal cells). Keep detection and exposure to chromogen consistent across the comparison so a stronger signal can be attributed to retrieval (standard IHC practice).
Could fixation explain weak or patchy ENO2 staining in my paraffin sections?
Target-specific sensitivity of ENO2 staining to fixation is unknown from the supplied evidence; the selected paraffin-section caption does not report a fixative (caption A02930). Record the fixative, fixation duration and tissue thickness for each specimen, then compare matched sections under the same citrate pH 6 retrieval and detection conditions (standard IHC practice; datasheet A02930). Uneven preservation can complicate interpretation of patchy chromogenic staining, so review morphology and include a known positive section in the same run (standard IHC practice; HPA tissue IHC: high in neuronal cells). Do not assign a fixation effect from ENO2 localisation or modification annotations alone (UniProt P09104).
Should ENO2 staining be cytoplasmic, membranous or nuclear?
Expect predominantly cytoplasmic staining in neuronal populations and pancreatic islets in tissue sections (HPA tissue IHC: cytoplasmic expression in CNS, peripheral nerves and pancreatic islets). ENO2 is annotated in the cytoplasm and at the cell membrane, despite having no transmembrane segment (UniProt P09104). A membrane accent can therefore be plausible, but assess it against cell morphology and a matched positive control before scoring it as specific (UniProt P09104; standard IHC practice). Predominantly nuclear signal does not fit the supplied localisation evidence; check counterstain, chromogen deposits and secondary-only control before treating it as ENO2 (HPA tissue IHC; UniProt P09104; standard IHC practice).
Can isoforms or epitope changes explain inconsistent ENO2 IHC staining?
The record lists 2 ENO2 isoforms and modified residues, but does not identify the catalog antibody’s epitope or establish isoform-specific recognition (UniProt P09104; caption A02930). Mammalian enolase subunits form cell-type-dependent dimers, including gamma-containing dimers in neurons, so cell composition matters when comparing sections (UniProt P09104). Do not infer that a particular modification masks this antibody’s epitope without direct validation, even though phosphorylation and acetylation are annotated (UniProt P09104). Compare staining in a documented positive neuronal region, review the antibody’s epitope information if available, and keep retrieval and detection matched across samples (HPA tissue IHC: high in neuronal cells; standard IHC practice).
How should I use IF to check an ambiguous ENO2 IHC pattern?
For a separate IF check, multiplex ENO2 with a validated neuronal marker and ask whether signal overlaps the expected cell population (HPA tissue IHC: high in neuronal cells; standard IF practice). Choose fluorophores and filters after inspecting unstained tissue autofluorescence, with a brighter, spectrally separated channel for a weak target signal (standard IF practice). Because ENO2 is cytosolic and can also localise to the plasma membrane, use permeabilisation when testing intracellular recognition, then compare with a minimally permeabilised condition if membrane signal is of interest (UniProt P09104; standard IF practice). The cited IF localisation includes a multi-gene antibody caution, so corroborate apparent membrane enrichment independently (HPA subcellular).
How can I distinguish ENO2 signal from brown background?
Run a no-primary control and inspect whether diffuse brown deposit persists, especially where endogenous peroxidase activity or nonspecific detection may contribute (standard chromogenic IHC practice). Include a peroxidase block and optimise the DAB development time as general workflow controls; the selected example used biotinylated secondary detection and DAB (standard chromogenic IHC practice; caption A02930). Its section was blocked with 10% goat serum, which provides a starting condition for this catalog antibody (caption A02930). Compare questionable areas with an expected positive neuronal region and inspect adjacent morphology before increasing primary concentration (HPA tissue IHC: high in neuronal cells; standard IHC practice).
What is a defensible way to quantify ENO2 staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored compartment and cell population before analysis: ENO2 is predominantly cytoplasmic in the reported tissue profile, with membrane localisation also annotated (HPA tissue IHC; UniProt P09104). For chromogenic sections, report an H-score based on staining intensity and percentage of positive cells, or report positive cells per mm² in a prespecified region (standard IHC practice). Normalise counts to viable tissue area or the number of eligible cells, and use identical retrieval, imaging and threshold rules across samples (standard IHC practice). Score neuronal and non-neuronal compartments separately where both are present, because reported ENO2 expression differs by cell type (HPA tissue IHC).
What would make an apparent ENO2-positive area unconvincing?
A convincing result follows viable cells with the expected cytoplasmic pattern and agrees with known neuronal staining in a positive control (HPA tissue IHC: cytoplasmic expression and high neuronal staining; standard IHC practice). Treat isolated nuclear colour, section-edge accentuation or staining confined to necrotic material cautiously, and compare those areas with morphology and a no-primary control (UniProt P09104; standard IHC practice). Persistent brown signal without primary antibody points to the detection workflow, including possible endogenous enzyme activity, rather than established ENO2 recognition (standard chromogenic IHC practice). Membrane-associated signal can be biologically plausible, but require cell-resolved evidence because ENO2 also localises to the plasma membrane (UniProt P09104; HPA subcellular).
Boster reagents
Best ENO2 / Gamma-enolase IHC Antibodies
Anti-ENO2 antibodies have IHC images from human and rodent paraffin sections (catalog IHC captions); IF/ICC images show A431 cells (catalog IF 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 shows IHC in human lung cancer, human placenta, rat brain and mouse brain, plus IF/ICC in A431 cells (A02930 image captions). PA1061 shows IHC in rat and human brain, while M02930-1 shows IHC in human uterus cancer and IF in A431 cells (PA1061 and M02930-1 image captions).
Which to pick: For paraffin-section IHC, choose A02930 for the broadest pictured tissue and species coverage; PA1061 has pictured rat and human brain, and M02930-1 has pictured human uterus cancer (each SKU’s IHC captions). For IF/ICC, choose A02930 or rabbit monoclonal M02930-1, both listed for IF/ICC with A431 images (catalog applications and IF captions); A02930 has pictured IHC examples in human, mouse and rat (A02930 IHC captions). The paraffin captions do not report a fixative (catalog 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); membrane translocation is possible (UniProt). 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: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Pancreatic endocrine cells stain less strongly than CNS neurons (HPA tissue IHC); Isoform / epitope: 2 isoforms; check epitope coverage across variants (UniProt); Regulation: Enolase dimers vary with 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, PMC8140915, PMC8253290. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ENO2/immunohistochemistry-ihc-guide.