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CEBPE / CCAAT/enhancer-binding protein epsilon · IHC design guide
Design Immunohistochemistry for CEBPE
Plan CEBPE chromogenic IHC around nuclear staining in bone marrow hematopoietic cells (HPA tissue IHC). The IHC-validated antibody A03884-1 has a starting dilution range of 1:100–1:300 (datasheet); include a no-primary control to assess chromogenic background (standard IHC practice).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CEBPE 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 in bone marrow hematopoietic cells (HPA tissue IHC) | |
| Staining pattern | Nuclear staining in bone marrow hematopoietic cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Bone marrow+1 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific) | |
| Caveat | Endogenous myeloid peroxidase may add background (HPA tissue IHC; standard IHC practice) | |
| Regulation | Strongest in promyelocyte-like cell lines (UniProt) | |
| Isoform / epitope | No isoforms or processing; one 1–281 chain (UniProt) |
Section 1
Recommended CEBPE IHC & IF Protocols
The catalog antibody protocol is presented alongside published CEBPE IHC methods for human cartilage endplate and rat tissues (PMC12268938) and tumor and normal tissues (PMC11375656).
| Sample | Paraffin-embedded human brain tissue; fixative not specified (datasheet A03884-1) |
| Fixation | Image fixative and duration unreported (datasheet A03884-1); 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 anti-CEBPE, 1:100-1:300 (datasheet A03884-1) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CEBPE-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: Nuclear expression in bone marrow poietic cells. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 at 95–98 °C for 20 min (page antigen-retrieval setting); one published method also specifies heat retrieval (PMC11375656 methods).
Section 2
What Is the Expected CEBPE Staining Pattern?
CEBPE should appear primarily in nuclei of bone marrow hematopoietic cells, where HPA reports high staining and an Enhanced tissue IHC reliability rating (HPA: High in bone marrow hematopoietic cells; Enhanced). Medium staining is reported in preleptotene spermatocytes (HPA: Medium in testis). A nuclear pattern fits this DNA-binding transcription factor, which has no transmembrane segment (UniProt Q15744: nucleus; no transmembrane segment).
| Clear nuclear chromogen in bone marrow hematopoietic cells, with surrounding tissue comparatively quiet. | This matches the principal tissue IHC pattern (HPA: nuclear expression in bone marrow poietic cells; High in hematopoietic cells). Interpret staining by cell and compartment, rather than treating every brown area as CEBPE (general IHC practice). |
| Medium nuclear staining in preleptotene spermatocytes. | This is another reported positive pattern (HPA: Medium in testis preleptotene spermatocytes). It need not match the intensity of the high bone marrow signal; compare like cells under the same staining conditions (general IHC practice). |
| Predominantly membranous or diffuse cytoplasmic chromogen in an IHC section. | This conflicts with the expected nuclear tissue pattern (HPA: nuclear expression in bone marrow poietic cells; UniProt Q15744: nucleus). Check morphology and controls before calling it specific; HPA's additional membrane and cytosol locations are uncertain ICC-IF observations (HPA: subcellular location). |
| Strong staining in cells reported as negative, such as adipocytes or bronchial respiratory epithelium. | These cells are listed as not detected (HPA: adipose adipocytes; bronchus respiratory epithelial cells). Consider cross-reactivity or endogenous detection activity, then compare a reagent control and an independently validated antibody (general IHC practice; HPA: two IHC Enhanced antibodies). |
| A widespread haze obscures nuclei, or known-positive bone marrow hematopoietic cells have no signal. | Neither gives an interpretable positive result (HPA: High in bone marrow hematopoietic cells; nuclear profile). Haze warrants background controls; absent signal warrants a check of retrieval, antibody and detection steps (general IHC practice). |
💡Expected CEBPE appearanceCall a result positive when discrete nuclear chromogen is high in bone marrow hematopoietic cells (HPA: High; nuclear profile); isolated diffuse cytoplasmic or membranous color is suspect (UniProt Q15744: nucleus; HPA: additional ICC-IF locations uncertain).
| Expected compartment | CEBPE is a nuclear DNA-binding transcription factor without a transmembrane segment (UniProt Q15744: nucleus; function; topology). Give nuclear staining priority when interpreting IHC; the uncertain additional ICC-IF locations do not establish a tissue IHC membrane pattern (HPA: subcellular location). |
| Choice of comparison tissue and cell | Bone marrow hematopoietic cells offer a reported high signal; preleptotene spermatocytes offer a reported medium signal (HPA: tissue IHC). Adipocytes and bronchial respiratory epithelial cells are reported as not detected and can help assess unexpected staining (HPA: tissue IHC). |
| Antibody evidence | HPA002928 and CAB005113 each have Enhanced IHC status (HPA: antibody validation). HPA describes its tissue IHC reliability as Enhanced because staining and RNA data show high consistency (HPA: tissue IHC). These ratings support the reported pattern; they do not specify a dilution or prove every stain in a new run is specific. |
| Antigen retrieval and fixation sensitivity | No CEBPE-specific fixation effect or retrieval condition is supplied (UniProt Q15744; HPA: tissue IHC). If signal needs optimization, compare retrieval conditions using reported positive cells and matched controls; treat this as general IHC practice, without inferring target-specific epitope masking. |
| Situation | Likely cause | Next action |
|---|---|---|
| Bone marrow section has little or no nuclear staining. | The expected high-positive cells are missing or a staining step underperformed (HPA: High in bone marrow hematopoietic cells; general IHC practice). | Confirm hematopoietic cells are present, then check antibody dilution, retrieval and detection with a positive control (general IHC practice). No CEBPE-specific retrieval setting is established here. |
| Chromogen spreads across the section and nuclear borders are hard to score. | Diffuse background may reflect nonspecific antibody binding or detection background (general IHC practice). | Review a reagent control, blocking, washes and detection exposure; score only resolved cellular staining (general IHC practice). Compare with the expected nuclear profile (HPA: tissue IHC). |
| Membrane or cytoplasm stains more strongly than nuclei. | The compartment differs from the tissue IHC expectation (HPA: nuclear bone marrow profile; UniProt Q15744: nucleus). | Check section morphology, reagent controls and staining with another IHC Enhanced antibody (general IHC practice; HPA: HPA002928 and CAB005113). Do not use uncertain ICC-IF membrane or cytosol calls to validate this IHC result (HPA: subcellular location). |
| Adipocytes or bronchial respiratory epithelial cells show prominent color. | Those cell types are reported as not detected (HPA: tissue IHC); cross-reactivity or endogenous detection activity is possible (general IHC practice). | Compare reagent controls, inspect the cellular compartment and repeat with an independently validated antibody if needed (general IHC practice; HPA: two IHC Enhanced antibodies). |
| Testis staining appears weaker than the bone marrow control. | This may reflect the reported difference between medium preleptotene spermatocyte staining and high bone marrow hematopoietic staining (HPA: tissue IHC). | Identify preleptotene spermatocytes and assess nuclear staining under matched conditions before treating lower intensity alone as assay failure (HPA: tissue IHC; general IHC practice). |
| Can an IF/ICC signal be read like the IHC result? | HPA supports nucleoplasmic ICC-IF localization, while its additional plasma membrane and cytosol assignments are uncertain (HPA: subcellular location). | Use nucleoplasmic signal as the main localization check and interpret extra-compartment IF signal cautiously (HPA: subcellular location). Follow the separate IF/ICC guide for its method; this section provides no IF protocol. |
Experiment evidence
IHC & IF images showing CEBPE staining
A03884-1 · IHCImmunohistochemistry analysis of paraffin-embedded human brain tissue, using C/EBP-epsilon Antibody. The picture on the right is blocked wit
A03884-1 · IFImmunofluorescence analysis of HeLa cells, using C/EBP-epsilon Antibody. The picture on the right is blocked with the synthesized peptide.
A03884-2 · IHCImmunohistochemistry (IHC) analyzes of C/EBP-ε (K70) pAb in paraffin-embedded human breast carcinoma tissue at 1:100.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000092067-CEBPE/tissue/cerebral+cortex#imid_992000
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000092067-CEBPE/tissue/colon#imid_991918
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000092067-CEBPE/tissue/liver#imid_991935
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000092067-CEBPE/tissue/kidney#imid_991928
HPA · ICC-IF JURKATHuman Protein Atlas immunofluorescence, JURKAT, antibody HPA002928; green = CEBPE, blue = nucleus, red = microtubules.
HPA · ICC-IF JURKATHuman Protein Atlas immunofluorescence, JURKAT, antibody HPA002928; green = CEBPE, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CEBPE IHC & IF
🧪Run bone marrow first and look for nuclear staining in hematopoietic cells (HPA: High in bone marrow hematopoietic cells; UniProt Q15744: nucleus). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes), and assess nonhematopoietic cells on the marrow slide as candidate internal negatives that should lack nuclear staining (HPA: High in hematopoietic cells; standard IHC practice).
Positive control tissue: Bone marrow (Hematopoietic cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CEBPE in JURKAT, REH, U2OS, A-431, U-251MG, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, an isotype control matched to the primary antibody’s host species and format, and either a validated CEBPE knockout sample or peptide competition (standard IHC practice; A03884-1 caption: peptide-blocked image). Quench endogenous peroxidase in bone marrow before chromogenic detection because myeloid cells can contribute background (standard IHC practice).
⚠️Feasibility: A CEBPE-specific fixation window or fixation effect is unreported, and the A03884-1 paraffin-section caption does not state a fixative (supplied target/application evidence; A03884-1 caption). Retrieval dependence is unreported; optimize antigen retrieval for paraffin IHC, and the supplied evidence does not establish whether frozen sections or IF/ICC are easier (standard IHC practice; supplied target/application evidence). For IF/ICC, prioritize nucleoplasmic staining and interpret membrane or cytosolic signal cautiously (HPA: nucleoplasm supported; plasma membrane and cytosol uncertain).
HPA tissue IHC evidence for CEBPE
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 |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Testis | Preleptotene spermatocytes | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CEBPE IHC Tips
Troubleshoot CEBPE staining in paraffin sections by checking nuclear signal, hematopoietic cell identity, and assay controls (UniProt Q15744; HPA tissue IHC).
How should I retrieve CEBPE in paraffin sections with weak nuclear staining?
Start with Tris-EDTA pH 9.0 HIER at 95–98 °C for 20 min (page retrieval rule: nuclear antigen). Keep section thickness, heating time, cooling, and detection conditions consistent while comparing runs, because each can change apparent nuclear intensity (standard IHC practice). Assess retrieval against hematopoietic cells in bone marrow, where high staining is reported, and inspect nuclei for both signal and preserved morphology (HPA tissue IHC; standard IHC practice). If staining remains weak, compare a citrate buffer at pH 6.0 as a fallback on matched sections, recording any gain in background or tissue damage (standard IHC practice).
Could fixation explain variable CEBPE staining between paraffin blocks?
Target-specific CEBPE sensitivity to fixation is unknown from the supplied evidence; the selected antibody caption identifies paraffin-embedded brain tissue but does not state its fixative (caption A03884-1). Record each block’s fixative, fixation duration, processing history, and section age before attributing differences to antigen abundance (standard IHC practice). Compare matched sections using the same Tris-EDTA pH 9.0, 95–98 °C, 20 min retrieval and identical detection settings (page retrieval rule; standard IHC practice). Include a bone marrow reference with hematopoietic cells in each run and judge staining alongside nuclear preservation and background (HPA tissue IHC; standard IHC practice).
What should I do if CEBPE staining appears mainly cytoplasmic?
Prioritize interpretable nuclear staining: CEBPE is annotated as nuclear, and its main supported subcellular location is the nucleoplasm (UniProt Q15744; HPA subcellular). HPA also lists cytosol and plasma membrane as uncertain additional locations, so isolated staining there needs independent confirmation (HPA subcellular). Compare the suspect area with a nuclear counterstain and with bone marrow hematopoietic cells, for which high tissue staining is reported (HPA tissue IHC; standard IHC practice). Recheck peroxidase blocking, primary antibody concentration, wash stringency, and section edges before calling diffuse cytoplasmic color a CEBPE pattern (standard IHC practice).
How can I assess whether the antibody recognizes the relevant CEBPE epitope?
Map the antibody’s documented immunogen or epitope against the 281-residue CEBPE sequence; the supplied record reports no isoforms (UniProt Q15744). The bZIP region spans residues 204–267, and phosphoserine is reported at residue 181, but neither fact identifies this antibody’s epitope (UniProt Q15744). Ask whether processing or retrieval could affect access to the documented epitope, then compare matched sections under identical staining conditions (standard IHC practice). The selected paraffin-section image includes synthesized-peptide blocking; reduced staining in that comparison supports competition with that peptide but cannot alone establish cell-type specificity (caption A03884-1; standard IHC interpretation).
How should I check a CEBPE signal by multiplex IF?
Treat IF as a separate assay requiring its own antibody validation and fixation assessment; the selected paraffin IHC caption does not report IF conditions (caption A03884-1; standard IF practice). Pair CEBPE with a validated hematopoietic or granulocytic lineage marker and a nuclear counterstain, since bone marrow hematopoietic cells show high staining and nucleoplasm is the main supported location (HPA tissue IHC; HPA subcellular). Choose fluorophores after measuring tissue autofluorescence and include single-color controls to identify bleed-through (standard IF practice). For an intracellular nuclear epitope, optimize detergent permeabilisation on matched samples, checking nuclear access and retention of the lineage-marker signal (UniProt Q15744; standard IF practice).
How can I distinguish CEBPE signal from diffuse brown background?
First compare cellular localization: convincing CEBPE signal should include nuclei, consistent with its nuclear annotation and supported nucleoplasmic location (UniProt Q15744; HPA subcellular). In chromogenic sections, check no-primary controls, endogenous peroxidase blocking, nonspecific binding blocks, and DAB development time on matched slides (standard IHC practice). Adjust primary antibody concentration and washing systematically, recording whether background falls without losing the expected bone marrow hematopoietic signal (HPA tissue IHC; standard IHC practice). Inspect pigment, folds, torn areas, and section edges under brightfield before treating brown deposits as positive cells (standard IHC practice). The selected peptide-blocked image offers a competition comparison, with the specificity limit described above (caption A03884-1).
What is a defensible way to quantify CEBPE IHC across samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region before scoring, then count counterstained nuclei and CEBPE-positive nuclei in comparable, viable areas (standard IHC practice). Report the percentage of positive cells and, if intensity grading is reproducible, an H-score using 0–3 intensity categories (standard IHC practice). Normalize positive counts to all eligible cells of the same type, or report positive-cell density per mm² when cellularity varies (standard IHC practice). Keep retrieval, detection, image exposure, thresholds, and exclusion rules constant, and include a bone marrow reference because hematopoietic cells have reported high staining (page retrieval rule; HPA tissue IHC; standard IHC practice).
When should I reject an apparent CEBPE-positive area as artefact?
Require signal in appropriate nuclei and cells: CEBPE is nuclear, while HPA reports high staining in bone marrow hematopoietic cells (UniProt Q15744; HPA tissue IHC). Treat dominant staining outside nuclei cautiously, since the reported cytosolic and plasma-membrane locations are uncertain (HPA subcellular). Exclude section-edge enhancement, folds, necrotic areas, pigment, and residual endogenous peroxidase before assigning chromogenic positivity (standard IHC practice). Compare suspicious fields with no-primary controls, a bone marrow reference, and the peptide-blocking comparison when relevant to this catalog antibody (HPA tissue IHC; caption A03884-1; standard IHC practice). Do not infer cell identity or disease state from brown color alone (standard IHC interpretation).
Boster reagents
Best CEBPE / CCAAT/enhancer-binding protein epsilon IHC Antibodies
Two anti-CEBPE antibodies have paraffin-section IHC images (catalog IHC captions); one also has a HeLa-cell IF image (A03884-1 IF caption). Both list human, mouse and rat reactivity (catalog).
Real IHC data
Anti-C/EBP epsilon CEBPE Antibody
Cat # A03884-1
Real IHC data
Anti-C/EBP-Epsilon (K70) CEBPE Antibody
Cat # A03884-2
A03884-1 will render with human brain paraffin-section IHC, including a peptide-blocked comparison; its catalog also lists IF and shows HeLa-cell IF (A03884-1 image captions; catalog). A03884-2 will render with human breast carcinoma paraffin-section IHC at 1:100; its catalog lists IF but supplies no IF image (A03884-2 IHC caption; catalog).
Which to pick: For tissue IHC, choose A03884-1 when a peptide-blocked IHC comparison is useful, or A03884-2 when the breast carcinoma example is relevant; each caption establishes paraffin sections, and neither reports the fixative (IHC image captions). For IF/ICC planning, A03884-1 has a HeLa-cell IF image, while A03884-2 only lists IF; neither payload establishes an ICC protocol (IF image captions; catalog applications). Both list human, mouse and rat reactivity, so neither has a stated species advantage; A03884-1 is labeled polyclonal, while A03884-2 has no explicit clonality field (catalog).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q15744 (CEBPE_HUMAN, CCAAT/enhancer-binding protein epsilon).
- Human Protein Atlas. CEBPE tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CEBPE subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the plasma membrane and cytosol..
- Human Protein Atlas. CEBPE antibody validation summary (2 antibodies).
- Deliver CEBPE via cartilage targeting Lipid nanoparticle to block CEBPE-LTF-STAT3 positive feedback loop for efficient treatment of cartilage endplate degeneration. Materials today. Bio 2025 — PMC12268938.
- Comprehensive Analysis of CCAAT/Enhancer Binding Protein Family in Ovarian Cancer. Cancer informatics 2024 — PMC11375656.
- PubMed PMID:8661101 — UniProt-cited evidence.
- PubMed PMID:9032264 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CEBPE (CCAAT/enhancer-binding protein epsilon). Expected localisation Nuclear in bone marrow hematopoietic cells (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A03884-1 at 1:100-1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Bone marrow (Hematopoietic cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human brain tissue; fixative not specified (datasheet A03884-1); 2. Fixation: Image fixative and duration unreported (datasheet A03884-1); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-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 anti-CEBPE, 1:100-1:300 (datasheet A03884-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific); Caveat: Endogenous myeloid peroxidase may add background (HPA tissue IHC; standard IHC practice); Isoform / epitope: No isoforms or processing; one 1–281 chain (UniProt); Regulation: Strongest in promyelocyte-like cell lines (UniProt). Evidence: UniProt Q15744, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A03884-1, PMC12268938, PMC11375656. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CEBPE/immunohistochemistry-ihc-guide.