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CCNE2 / G1/S-specific cyclin-E2 · IHC design guide
Design Immunohistochemistry for CCNE2
Plan CCNE2 chromogenic IHC in paraffin sections around its nuclear staining pattern, which appears in a varying fraction of tissue cells (HPA tissue IHC). This guide covers fixation, staining controls and scoring of positive nuclei.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CCNE2 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 | Nuclear staining in a varying fraction of cells (HPA tissue IHC) | |
| Staining pattern | Nuclear staining in subsets of glandular and hematopoietic cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Appendix+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). Selected-image fixative and duration unreported (datasheet M04536); verify before use. | |
| Caveat | The fraction of positive nuclei varies between tissues (HPA tissue IHC) | |
| Regulation | Elevated in tumor-derived cells (UniProt) | |
| Isoform / epitope | 2 isoforms, Long and Short; epitope coverage is unknown (UniProt) |
Section 1
Recommended CCNE2 IHC & IF Protocols
The catalog antibody IHC-P protocol is accompanied by published CCNE2 protocols for paraffin sections (PMC8459753; PMC8656552).
| Sample | Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet M04536) |
| Fixation | Image fixative and duration unreported (datasheet M04536); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit monoclonal (clone GHH-3) anti-CCNE2, 1:50 (datasheet M04536) |
| 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 | CCNE2-positive staining in glandular cells of appendix (HPA tissue IHC: Medium). HPA tissue profile: Nuclear expression in varying fraction of cells in several tissues. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA HIER at pH 9.0, 95–98 °C for 20 min (page retrieval); a published protocol also uses EDTA at pH 9.0 (PMC8656552).
Section 2
What Is the Expected CCNE2 Staining Pattern?
CCNE2 should appear primarily in nuclei, in a varying fraction of cells rather than uniformly across a section (UniProt O96020: nucleus; HPA tissue IHC: nuclear expression). HPA reports medium staining in selected glandular, hematopoietic, glial, germinal center, trophoblastic and keratinocyte populations (HPA tissue IHC). Its tissue assessment is Enhanced, with medium consistency between antibody staining and RNA expression (HPA tissue IHC). CCNE2 has no transmembrane segment (UniProt O96020: topology).
| Discrete nuclear staining in some hematopoietic cells of bone marrow or germinal center cells of lymph node, with other cells unstained. | This fits the reported medium staining in those populations and the varying fraction of nuclear-positive cells (HPA tissue IHC). Record the positive fraction and intensity within the identified cell population; a uniformly positive field is not required by the HPA pattern (HPA tissue IHC). |
| Predominantly cytoplasmic or membrane-like staining, with little nuclear signal. | This conflicts with the recorded nuclear location and absence of a transmembrane segment (UniProt O96020: location and topology; HPA tissue IHC). Treat it as a possible staining artefact, then review morphology and controls before assigning a CCNE2 result (general IHC practice). |
| Strong staining in adipocytes or in an unrelated cell population while expected nuclei are negative. | HPA reports adipocytes as not detected in its listed adipose and breast samples (HPA tissue IHC). Check whether the signal follows tissue pigments, endogenous detection activity or antibody cross-reactivity; morphology and control sections help distinguish these possibilities (general IHC practice). |
| Diffuse color across tissue, including extracellular spaces, with no clear nuclear boundaries. | This does not resemble the reported nuclear pattern (HPA tissue IHC; UniProt O96020: nucleus). Review no-primary and detection controls, blocking, washing and chromogen development before interpreting weak nuclear color against the background (general IHC practice). |
| No signal in a section chosen as a positive comparator. | First confirm that the sampled cell population matches a reported positive one: HPA lists medium staining in bone marrow hematopoietic cells, not every cell in bone marrow (HPA tissue IHC). A negative field can reflect sampling or a staining-run problem; compare the control slide and run conditions before calling the specimen negative (general IHC practice). |
💡Expected CCNE2 appearanceCall a result consistent with CCNE2 when distinct nuclei in a subset of HPA-reported cell populations show interpretable staining, including the reported medium intensity in selected populations; isolated diffuse, cytoplasmic or membrane-like color is suspect (HPA tissue IHC; UniProt O96020: location and topology).
| Cell-cycle distribution | CCNE2 acts in late G1 and early S phase (UniProt O96020: function), and HPA reports a varying fraction of nuclear-positive cells (HPA tissue IHC). Interpret the fraction within each cell population rather than expecting every nucleus to stain. |
| Choice of tissue and cell population | HPA reports medium staining in appendix glandular cells, placenta trophoblastic cells and skin keratinocytes, but not detected staining in several other specified cell populations (HPA tissue IHC). Name the cell population when selecting and scoring a comparator. |
| Antibody evidence | CAB007825 has Enhanced IHC status and CAB019374 has Approved IHC status (HPA antibodies). The HPA tissue profile has Enhanced reliability with medium RNA–staining consistency (HPA tissue IHC); these ratings do not establish that every specimen or cell will stain. |
| Isoforms and epitope | UniProt lists Long and Short isoforms and a single annotated 1–404 chain (UniProt O96020: isoforms and processing). The supplied records give no antibody epitope or isoform coverage, so isoform-specific staining cannot be assigned from these data. |
| IF/ICC: is the nuclear pattern independently confirmed by cell images? | HPA supplies no main ICC-IF location, additional location or cell-line images for CCNE2 (HPA subcellular). Use the IHC nuclear pattern as an expectation only; this record cannot establish an IF/ICC staining pattern or protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| A putative positive section shows no nuclear staining. | The examined field may lack the relevant cells, or the staining run may have failed (HPA tissue IHC: varying positive fraction; general IHC practice). | Locate the reported cell population on the section, then inspect the positive control and the run's reagent and detection records before interpreting absence of signal (HPA tissue IHC; general IHC practice). |
| Color is concentrated outside nuclei. | The distribution disagrees with CCNE2's recorded nuclear location (UniProt O96020: nucleus; HPA tissue IHC). Nonspecific staining or misidentified cell boundaries are possible (general IHC practice). | Review the counterstain and morphology, compare a no-primary control, and score only clearly assigned nuclei (general IHC practice). |
| Many unexpected cells stain, including adipocytes. | HPA lists adipocytes as not detected in adipose tissue and breast samples (HPA tissue IHC). Unexpected color may arise from cross-reactivity or endogenous detection activity (general IHC practice). | Check the affected cell identity and control sections; compare staining with the reported positive and negative populations before attributing the color to CCNE2 (HPA tissue IHC; general IHC practice). |
| Weak nuclear color cannot be separated from diffuse background. | Background from the detection workflow can obscure a varying fraction of positive nuclei (general IHC practice; HPA tissue IHC: nuclear expression in varying fractions). | Inspect no-primary and detection controls; review blocking, washes and chromogen development, then reassess whether individual nuclei have distinct signal (general IHC practice). |
| A low-staining lung sample is treated as a failed positive control. | HPA reports low staining in lung alveolar cells, while UniProt cites elevated lung expression from one literature account (HPA tissue IHC; UniProt O96020: tissue specificity). The records address different evidence and do not guarantee strong IHC staining. | Use the HPA cell-specific IHC observation to judge that slide; choose a reported medium-staining population when a clearer IHC comparator is needed (HPA tissue IHC). |
| Two sections differ in the fraction of stained nuclei. | HPA describes a varying positive fraction, and CCNE2 has a late-G1/early-S cell-cycle role (HPA tissue IHC; UniProt O96020: function). Neither source establishes a target-specific fixation effect. | Compare the same identified cell population, record positive fraction and intensity, and check run controls before treating the difference as biological (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing CCNE2 staining
M04536 · IHCImmunohistochemical analysis of paraffin-embedded human breast carcinoma, using Cyclin E2 Antibody.
M04536 · IFImmunofluorescent analysis of Hela cells, using Cyclin E2 Antibody.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000175305-CCNE2/tissue/cerebral+cortex#imid_2350264
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000175305-CCNE2/tissue/colon#imid_2350174
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000175305-CCNE2/tissue/liver#imid_2350191
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000175305-CCNE2/tissue/kidney#imid_2350192
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CCNE2 IHC & IF
🧪Run appendix first: glandular cells should show CCNE2 staining (HPA: Medium in appendix glandular cells). Use adipose tissue as the biological negative (HPA: Not detected in adipocytes); on the appendix slide, adjacent cells without nuclear signal provide an internal background reference, without assuming every nonglandular cell is negative (UniProt O96020: nucleus).
Positive control tissue: Appendix (Glandular cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for CCNE2; derive a cell-line control from the positive tissue's cell type (Glandular cells) and confirm it by RNA or western blot first.
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). A CCNE2 knockout specimen, if available, provides a target-specific biological negative; quench endogenous peroxidase in the appendix section before chromogenic detection and check for endogenous biotin if using avidin–biotin detection (standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence, and the fixative for the selected paraffin-section breast carcinoma image is unreported (M04536 caption: fixative not stated). Antigen retrieval dependence is unreported, so optimize retrieval with the catalog antibody’s IHC-P protocol (standard IHC practice). There is no supplied evidence that frozen sections or IF are easier for CCNE2; in appendix, distinguish nuclear signal from background due to endogenous peroxidase (UniProt O96020: nucleus; standard IHC practice).
HPA tissue IHC evidence for CCNE2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium 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 |
|---|---|---|---|---|
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Caudate | Glial cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | Cells in molecular layer | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | Glial 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 → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CCNE2 IHC Tips
Troubleshoot CCNE2 staining in paraffin sections by checking retrieval, nuclear localisation, antibody specificity and cell level scoring before interpreting chromogenic signal.
How should I retrieve CCNE2 when nuclear staining is weak?
Use Tris-EDTA pH 9.0 heat retrieval at 95–98 °C for 20 min (page retrieval rule: nuclear antigen). Apply the same cooling and wash conditions across sections, then compare nuclear signal with a no-primary control (standard IHC practice). If staining remains weak, test a shorter or longer heating interval on adjacent sections while holding antibody concentration and detection constant (standard IHC practice). Assess both signal and tissue integrity: excessive heating can damage sections or increase background (standard IHC practice). Score nuclei in comparable cell populations because CCNE2 expression varies with cell cycle phase (UniProt O96020: late G1 and early S function).
Could fixation explain absent CCNE2 staining in my paraffin sections?
Target-specific fixation sensitivity is unknown from the supplied evidence; the M04536 image identifies paraffin-embedded breast carcinoma but reports no fixative (M04536 tissue-IHC caption). Record the fixative and processing history for every specimen before comparing staining (standard IHC practice). For a controlled check, process matched samples with documented fixation intervals and stain them together using Tris-EDTA pH 9.0 retrieval for 20 min (page retrieval rule). Compare nuclear staining, morphology and background without assuming that any difference reflects a known CCNE2 fixation effect (standard IHC practice). Include a consistent reference section in each run to distinguish specimen processing differences from staining variability (standard IHC practice).
Where should convincing CCNE2 staining appear in chromogenic IHC?
Prioritise nuclear staining because CCNE2 is annotated in the nucleus and has no transmembrane segment (UniProt O96020: localisation and topology). Examine individual nuclei at high magnification, using the counterstain to separate nuclear DAB signal from adjacent cytoplasm (standard IHC practice). A varying fraction of stained cells is plausible: HPA reports nuclear expression in varying fractions across several tissues (HPA tissue IHC). Compare suspected positive cells with a no-primary control and with expected cell populations, such as lymph node germinal center cells (HPA tissue IHC: medium staining). Do not classify diffuse cytoplasmic colour alone as confirmed CCNE2; investigate nonspecific detection and section artefacts first (UniProt O96020: nucleus; standard IHC practice).
How can isoforms or epitope accessibility complicate CCNE2 IHC?
CCNE2 has Long and Short isoforms, but the supplied evidence does not identify the catalog antibody’s epitope or establish recognition of both (UniProt O96020: two isoforms). Obtain the immunogen or epitope location before assigning a negative section to loss of a particular isoform (standard antibody validation practice). CCNE2 also has annotated modified residues, including phosphoserines at 21 and 383 and phosphothreonine at 392 (UniProt O96020: modified residues). An epitope overlapping a modified or alternatively spliced region could alter recognition; treat that as a testable possibility, not an established cause (standard immunostaining practice). Compare retrieval conditions and independent specificity controls before interpreting altered nuclear intensity biologically (standard IHC practice).
How should I check CCNE2 localisation by IF alongside this IHC guide?
For the separate IF/ICC workflow, test nuclear access because CCNE2 is nuclear and lacks a transmembrane segment (UniProt O96020: localisation and topology). After fixation, compare a mild permeabilisation condition, such as 0.1% Triton X-100 for 5–10 min, with the workflow’s usual condition while monitoring morphology (standard IF practice). Multiplex with a marker identifying the expected cell population and include a nuclear counterstain so CCNE2 signal can be assigned to the correct cells (standard IF practice). Choose fluorophores in channels with low measured tissue autofluorescence and include single-colour and no-primary controls (standard IF practice). The paraffin-section M04536 caption supplies no IF/ICC validation or fixation condition (M04536 tissue-IHC caption).
What should I change when DAB obscures CCNE2-positive nuclei?
First inspect no-primary and reagent controls to distinguish antibody background from endogenous enzyme signal (standard chromogenic IHC practice). Use a peroxidase block before HRP/DAB detection and check whether tissue pigment or precipitate persists without primary antibody (standard chromogenic IHC practice). Titrate the catalog antibody across a small dilution series while keeping retrieval at Tris-EDTA pH 9.0, 95–98 °C for 20 min (page retrieval rule; standard IHC practice). Increase washing and reassess blocking if diffuse colour remains, then shorten chromogen development only after confirming the detection system works (standard IHC practice). Judge improvement by cleaner nuclei and preserved tissue morphology, not by overall darkness (UniProt O96020: nuclear localisation; standard IHC practice).
How should I score heterogeneous CCNE2 nuclear staining? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and count only evaluable nuclei, since HPA describes nuclear CCNE2 in varying fractions of cells (HPA tissue IHC). Report the percentage of positive nuclei and, when intensity is reproducible, an H-score calculated from 0–3 intensity categories, with a possible range of 0–300 (standard IHC scoring practice). Use the same positivity threshold, counterstain and imaging settings across cases, with blinded scoring when feasible (standard IHC practice). Normalise positive cell counts or densities to the number of evaluable target cells or sampled tissue area in mm², as appropriate (standard IHC practice). Exclude necrotic and damaged regions consistently, and report regional variation rather than hiding it in one average (standard IHC practice).
How can I distinguish real CCNE2 signal from section artefacts?
A credible result shows interpretable nuclear staining in an identifiable cell population, consistent with CCNE2’s nuclear annotation and HPA’s variable nuclear pattern (UniProt O96020: localisation; HPA tissue IHC). Compare suspicious staining with adjacent intact cells, a no-primary control and a reference section processed in the same run (standard IHC practice). Treat diffuse cytoplasmic colour, section-edge accentuation, necrotic deposits and signal restricted to endogenous enzyme-rich areas as possible artefacts requiring control checks (standard chromogenic IHC practice). Do not infer that every unstained cell lacks CCNE2, because its function is tied to late G1 and early S (UniProt O96020: cell cycle function). Record compartment, cell identity and positive-cell fraction before drawing biological conclusions (standard IHC practice).
Boster reagents
Best CCNE2 / G1/S-specific cyclin-E2 IHC Antibodies
Anti-CCNE2 antibodies cover IHC and IF/ICC in human and mouse samples (catalog: M04536); a rat-reactive option is also listed (catalog: A04536-3). M04536 has IHC and IF image captions (catalog: M04536).
Real IHC data
Anti-Cyclin E2 CCNE2 Rabbit Monoclonal Antibody
Cat # M04536
The rendered card is M04536, with IHC shown in paraffin-embedded human breast carcinoma (M04536 IHC image caption). It also lists IF/ICC for human and mouse and has an IF image caption for HeLa cells (catalog: M04536 applications, reactivity and IF image caption).
Which to pick: For tissue IHC, choose M04536: its own image caption documents paraffin-embedded human breast carcinoma, while the fixative is unreported (M04536 IHC image caption). For IF/ICC, M04536 lists both applications and has an IF image caption for HeLa cells (catalog: M04536 applications and IF image caption). For rat samples, consider polyclonal A04536-3, which lists rat reactivity and IHC and IF applications (catalog: A04536-3); its payload provides no IHC image caption.
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O96020 (CCNE2_HUMAN, G1/S-specific cyclin-E2).
- Human Protein Atlas. CCNE2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CCNE2 subcellular location (ICC-IF): Variation in transcript expression is correlated to the cell cycle..
- Human Protein Atlas. CCNE2 antibody validation summary (2 antibodies).
- Dystrophin-glycoprotein complex sequesters Yap to inhibit cardiomyocyte proliferation. Nature 2017 — PMC5528853.
- Circular RNA hsa_circ_0000073 Enhances Osteosarcoma Cells Malignant Behavior by Sponging miR-1252-5p and Modulating CCNE2 and MDM2. Frontiers in cell and developmental biology 2021 — PMC8459753.
- A Patient-Derived Xenograft Model of Dedifferentiated Endometrial Carcinoma: A Proof-of-Concept Study for the Identification of New Molecularly Informed Treatment Approaches. Cancers 2021 — PMC8656552.
- Self-organizing three-dimensional dermal papilla cell spheroids yield therapeutic extracellular vesicles that target hypertrophic scar regression via the miR-26a-5p/CCNE2 axis. Burns & trauma 2026 — PMC13345373.
- PubMed PMID:9858585 — UniProt-cited evidence.
- PubMed PMID:9840927 — UniProt-cited evidence.
- PubMed PMID:9840943 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CCNE2 (G1/S-specific cyclin-E2). Expected localisation Nuclear staining in a varying fraction of cells (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: M04536 at 1:50 (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet M04536); 2. Fixation: Image fixative and duration unreported (datasheet M04536); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit monoclonal (clone GHH-3) anti-CCNE2, 1:50 (datasheet M04536); 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 M04536); verify before use.; Caveat: The fraction of positive nuclei varies between tissues (HPA tissue IHC); Isoform / epitope: 2 isoforms, Long and Short; epitope coverage is unknown (UniProt); Regulation: Elevated in tumor-derived cells (UniProt). Evidence: UniProt O96020, HPA tissue IHC · Enhanced, HPA cell-line RNA · Variation in transcript expression is correlated to the cell cycle., Datasheet M04536, PMC8459753, PMC8656552. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CCNE2/immunohistochemistry-ihc-guide.