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EZH2 / Histone-lysine N-methyltransferase EZH2 · IHC design guide
Design Immunohistochemistry for EZH2
Plan EZH2 chromogenic IHC around the nuclear tissue pattern (HPA tissue IHC). Tonsil germinal center cells provide a high-staining reference (HPA tissue IHC); compare staining by cell type and score nuclei consistently.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
EZH2 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 several tissues (HPA tissue IHC) | |
| Staining pattern | Strong nuclear staining in spermatocytes and germinal center cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Bone marrow+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 paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining varies by cell type within tissues (HPA tissue IHC) | |
| Regulation | Overexpressed in several tumor types (UniProt) | |
| Isoform / epitope | 5 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended EZH2 IHC & IF Protocols
The catalog antibody protocol is followed by 3 published EZH2 IHC protocols for cervical carcinoma, bone marrow biopsies, and lung carcinoma.
| Sample | Paraffin-embedded human tonsil tissue; fixative not specified (datasheet M00050-1) |
| Fixation | Image fixative and duration unreported (datasheet M00050-1); 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 monoclonal (clone IDF-5) anti-EZH2, 1:50-1:200 (datasheet M00050-1) |
| 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 | EZH2-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: Nuclear expression in several tissues, most abundant in testis and lymphoid tissues. No signal in the no-primary control. |
💡Decision noteStart with the page’s Tris-EDTA pH 9.0 retrieval for the catalog antibody; the published protocols below report citrate pH 6.0 where retrieval is specified.
Section 2
What Is the Expected EZH2 Staining Pattern?
EZH2 should stain nuclei in paraffin-section IHC: UniProt places it in the nucleus and reports no transmembrane segment (UniProt Q15910). HPA reports nuclear expression across several tissues, strongest overall in testis and lymphoid tissues, with high staining in defined cell populations such as tonsil germinal center cells and testis pachytene spermatocytes (HPA tissue IHC). HPA rates its tissue profile Enhanced, while noting medium consistency between antibody staining and RNA expression (HPA tissue IHC).
| Distinct nuclear staining in tonsil germinal center cells or testis pachytene spermatocytes. | This matches two HPA populations scored High (HPA tissue IHC). Evaluate staining in the named cells, rather than treating the whole tissue section as uniformly positive. Nuclear localization also agrees with UniProt (UniProt Q15910). |
| Predominantly cytoplasmic or membranous staining, with little nuclear signal. | That distribution conflicts with EZH2's reported nuclear location and its nucleoplasmic IF location (UniProt Q15910; HPA subcellular). Treat it as a possible localization artefact; compare a known-positive nuclear pattern and a no-primary control before scoring it as EZH2. |
| Strong staining in adipocytes or cardiomyocytes instead of the expected positive cells. | HPA scores adipocytes in adipose tissue and cardiomyocytes in heart muscle Not detected (HPA tissue IHC). Unexpected staining may reflect cross-reactivity or endogenous chromogen-generating activity (standard IHC practice). Check the exact cell identity and a no-primary control; these HPA observations do not prove universal absence. |
| Colour spread across nuclei, cytoplasm and surrounding tissue without clear cell boundaries. | Diffuse signal cannot be confidently assigned to EZH2-positive nuclei (UniProt Q15910; standard IHC interpretation). Excess primary or detection reagent, incomplete blocking, or residual chromogen-generating activity are general IHC possibilities (standard IHC practice); compare background controls before changing the biological interpretation. |
| No nuclear signal in a section containing tonsil germinal center cells or testis pachytene spermatocytes. | These cell populations are scored High by HPA, so an entirely blank result warrants a technical check (HPA tissue IHC). Confirm that the expected cells are present, then review the antibody's IHC-P use, retrieval, dilution and detection controls (standard IHC practice). HPA supplies no EZH2-specific fixation sensitivity. |
💡Expected EZH2 appearanceCall a result positive when high signal is confined mainly to nuclei of HPA-high cells, such as tonsil germinal center cells, while broad cytoplasmic colour or strong staining in HPA-undetected adipocytes argues for a false-positive assessment (HPA tissue IHC; UniProt Q15910).
| Compartment and cell identity | EZH2 is nuclear (UniProt Q15910), and HPA IF places it in the nucleoplasm (HPA subcellular). For IHC, score nuclear signal within the specified cell population; staining elsewhere in the same tissue does not substitute for that observation (standard IHC interpretation). |
| Choice of tissue comparator | HPA scores bone marrow hematopoietic cells, placental cytotrophoblasts, skin granular-layer cells, testis pachytene spermatocytes and tonsil germinal center cells High (HPA tissue IHC). It scores several other populations Medium or Low; a weaker field can therefore reflect cell selection (HPA tissue IHC). |
| Antibody validation | HPA lists CAB009589 as IHC Enhanced and HPA029131 without an IHC validation status (HPA antibodies). The former offers an HPA-validated pattern comparator; the latter's ICC Supported status does not establish IHC-P performance (HPA antibodies). Check validation for the antibody actually used. |
| Isoforms and epitope coverage | UniProt lists five EZH2 isoforms (UniProt Q15910). If antibodies disagree, check their documented immunogens or epitope coverage before invoking isoform differences (standard IHC interpretation); these records provide no antibody-specific epitope map or isoform staining pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive tissue is blank. | The positive cell population may be absent from the examined field, or the IHC run may have failed (HPA tissue IHC; standard IHC practice). | Locate HPA-high cells, then check the IHC-validated antibody's stated application, retrieval instructions and working dilution alongside detection controls (HPA tissue IHC; standard IHC practice). |
| Signal appears mainly outside nuclei. | The result disagrees with nuclear EZH2 localization (UniProt Q15910); nonspecific staining or an interpretation error is possible (standard IHC practice). | Compare with a known-positive nuclear field and a no-primary control; assess nuclear boundaries with the counterstain before assigning positivity (standard IHC practice). |
| Strong colour appears in HPA-undetected cells. | Adipocytes and cardiomyocytes are reported Not detected in their specified tissues (HPA tissue IHC); cross-reactivity or endogenous detection activity is possible (standard IHC practice). | Verify cell identity and examine a no-primary control. If background remains, review the detection and blocking steps (standard IHC practice). |
| The whole section has diffuse colour. | Broad background can obscure a nuclear pattern; excess reagent or incomplete blocking are general possibilities (standard IHC practice). | Compare background controls, then review blocking, primary dilution, wash steps and chromogen development against the established IHC procedure (standard IHC practice). |
| Two antibodies give different IHC patterns. | HPA records IHC Enhanced for CAB009589 but no IHC status for HPA029131 (HPA antibodies); antibody performance and epitope coverage may differ. | Prioritize the antibody with documented IHC validation for pattern comparison and inspect each antibody's documented immunogen and application; do not infer an isoform-specific pattern from these records (HPA antibodies; UniProt Q15910). |
| For IF/ICC, where should EZH2 fluorescence appear? | HPA reports enhanced nucleoplasmic localization in ICC-IF, consistent with UniProt's nuclear annotation (HPA subcellular; UniProt Q15910). | Expect nucleoplasmic fluorescence and assess it with a nuclear counterstain (HPA subcellular; standard IF practice). Use the separate IF/ICC guide for its workflow; the IHC-P pattern alone does not validate an IF procedure. |
Experiment evidence
IHC & IF images showing EZH2 staining
M00050-1 · IHCImmunohistochemical analysis of paraffin-embedded human tonsil, using KMT6 / EZH2 Antibody.
M00050-1 · IFImmunofluorescent analysis of Hela cells, using KMT6 / EZH2 Antibody.
M00050-2 · IHCHumantonsil was stained with anti-EZH2 rabbit antibody
A00050 · IFImmunocytochemistry of EZH2 in 293 cells with EZH2 antibody at 10 μg/mL.
A00050-3 · IFImmunofluorescent analysis of 4% paraformaldehyde-fixed, 0. 1% Triton X-100 permeabilized Hela (Human cervical epithelial adenocarcinoma cel
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000106462-EZH2/tissue/cerebral+cortex#imid_2646548
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000106462-EZH2/tissue/colon#imid_2646482
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000106462-EZH2/tissue/liver#imid_2646467
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000106462-EZH2/tissue/kidney#imid_2646468
HPA · ICC-IF REHHuman Protein Atlas immunofluorescence, REH, antibody HPA029131; green = EZH2, blue = nucleus, red = microtubules.
HPA · ICC-IF REHHuman Protein Atlas immunofluorescence, REH, antibody HPA029131; green = EZH2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for EZH2 IHC & IF
🧪Run tonsil first: germinal center cells should show strong EZH2 staining (HPA: High in tonsil germinal center cells; selected-SKU caption: paraffin-embedded human tonsil). Run adipose tissue as the negative comparator, scoring adipocytes (HPA: Not detected in adipocytes). On the tonsil slide, cells outside the germinal centers should serve as a background reference, but their EZH2 status must be checked rather than assumed negative (HPA: High is specified for germinal center cells only).
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 EZH2 in REH, SiHa, U2OS, A-431, U-251MG, RPTEC/TERT1, hTERT-RPE1 (serum starved), KOLF2.1J, with annotated localisation: Nucleoplasm (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species, clonality and concentration; compare with EZH2 knockout material where available (standard IHC control practice). For chromogenic IHC, quench endogenous peroxidase and check the tonsil control slide for residual signal (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence, and the fixative for the selected-SKU tonsil caption is unreported (selected-SKU caption: paraffin-embedded human tonsil; fixative not stated). Antigen retrieval requirements and conditions are unreported, so assess retrieval empirically with the catalog antibody; the paraffin-section caption supports an IHC example, while the supplied HPA ICC-IF images support nucleoplasmic localisation, without establishing that frozen sections or IF are easier (selected-SKU caption: paraffin-embedded human tonsil; HPA subcellular: nucleoplasm). Check tonsil for residual endogenous peroxidase signal during chromogenic detection (standard IHC practice).
HPA tissue IHC evidence for EZH2
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 |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Placenta | Cytotrophoblasts | High | Protein (IHC) | HPA → |
| Skin | Cells in granular layer | High | Protein (IHC) | HPA → |
| Testis | Pachytene spermatocytes | High | Protein (IHC) | HPA → |
| Tonsil | Germinal center cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cerebral cortex | Endothelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced EZH2 IHC Tips
Troubleshoot EZH2 staining by checking nuclear localisation, cell type, tissue processing and controls before comparing signal across sections.
How should I adjust retrieval when EZH2 nuclear staining is weak?
Start with Tris-EDTA pH 9.0 heat-induced retrieval at 95–98 °C for 20 min (page retrieval specification). If germinal center nuclei in tonsil remain faint, check section adhesion, heating uniformity and primary-antibody exposure before changing retrieval; these cells provide a high-expression reference (HPA: High in tonsil germinal center cells). As a fallback, compare a shorter retrieval time or a lower-pH buffer on adjacent sections while keeping detection conditions fixed, then assess nuclear signal against tissue damage (standard IHC practice; UniProt Q15910: Nucleus). Record the retrieval condition for every section because variable heating can mimic biological differences in EZH2 staining (standard IHC practice).
Could fixation explain absent EZH2 staining in paraffin sections?
Target-specific fixation sensitivity is unknown because no EZH2 fixation comparison is supplied, and the catalog antibody’s paraffin-embedded tonsil caption does not state a fixative (selected IHC caption: M00050-1). Check the specimen’s recorded fixative and fixation duration, then compare sections processed together before attributing a weak result to EZH2 biology (standard IHC practice). Inspect nuclear preservation and tissue morphology; poorly preserved nuclei make a nuclear readout difficult to interpret (UniProt Q15910: Nucleus; standard IHC practice). Keep retrieval, antibody exposure and chromogenic development matched while testing processing differences, with tonsil germinal center cells as a reference population (HPA: High in tonsil germinal center cells).
What should I check when EZH2 staining appears mainly cytoplasmic?
Expect predominantly nuclear staining in paraffin sections because EZH2 is reported in the nucleus and its enhanced subcellular location is the nucleoplasm (UniProt Q15910: Nucleus; HPA: Nucleoplasm). Compare cytoplasmic color with adjacent nuclei and with a no-primary control before assigning the signal to EZH2 (standard IHC practice). Recheck focus, hematoxylin counterstain and chromogen development so a dense nuclear deposit is not mistaken for surrounding cytoplasm (standard IHC practice). Strong nuclear staining in tonsil germinal center cells offers a reference pattern, while persistent cytoplasmic staining warrants antibody-specific validation before biological interpretation (HPA: High in tonsil germinal center cells; standard IHC practice).
Can epitope choice explain differences between EZH2 IHC antibodies?
EZH2 has 5 listed isoforms and contains CXC residues 503–605 and SET residues 612–727, so document which sequence the antibody recognizes before comparing reagents (UniProt Q15910: Isoforms; Domains). The supplied evidence does not locate this catalog antibody’s epitope, so its isoform coverage and sensitivity to nearby modifications cannot be assigned (provided record: no antibody epitope specified). Compare staining on adjacent sections using the same retrieval and detection conditions, and record any differences in nuclear distribution by cell type (standard IHC practice; UniProt Q15910: Nucleus). For a discrepant pattern, seek mapped-epitope or orthogonal validation rather than inferring an isoform from stain intensity alone (standard IHC practice).
How can I assess EZH2 in a multiplex IF experiment?
For the separate IF/ICC workflow, pair EZH2 with a marker identifying the cell population of interest and a nuclear counterstain, then evaluate EZH2 within segmented nuclei (UniProt Q15910: Nucleus; standard IF practice). Choose fluorophores after checking the specimen’s autofluorescence in each channel, reserving a spectrally distinct, cleaner channel for weak EZH2 signal (standard IF practice). EZH2 has no transmembrane segment and is nucleoplasmic, so permeabilisation must allow antibody access across cellular and nuclear barriers; optimise it against nuclear morphology and background (UniProt Q15910: Topology; HPA: Nucleoplasm; standard IF practice). Use single-stain and secondary-only controls to identify bleed-through and nonspecific fluorescence before interpreting colocalisation (standard IF practice).
How do I reduce diffuse brown background without losing nuclear EZH2 signal?
Compare the stained section with no-primary and detection-only controls to locate background introduced by the primary antibody or chromogenic reagents (standard IHC practice). For peroxidase-based detection, check the endogenous-peroxidase block, protein block, wash steps and DAB development time; adjust one factor at a time while retaining nuclear signal (standard IHC practice). Examine adipocytes as a low-signal internal comparator where available, since EZH2 was not detected in adipocytes in the listed tissues (HPA: Not detected in adipose and breast adipocytes). Score nuclear deposits separately from diffuse stromal color, tissue folds and precipitate, and confirm that counterstaining still permits reliable nuclear identification (UniProt Q15910: Nucleus; standard IHC practice).
How should I quantify EZH2 across sections with different cell compositions? ⚠ ANSWER MARKED FOR VERIFICATION
Define a cell population and nuclear threshold before scoring, because EZH2 abundance varies by tissue and cell type (HPA: Tissue IHC profile; UniProt Q15910: Nucleus). Report the percentage of positive nuclei or an H-score based on nuclear intensity and positive-cell fraction, with the number of evaluable nuclei recorded for each region (standard IHC practice). For spatial comparisons, report positive nuclei per mm² and normalise to total evaluable nuclei or the relevant cell population in that area (standard IHC practice). Keep retrieval, imaging and scoring thresholds consistent, and exclude folds, necrosis and poorly preserved regions before comparing specimens (standard IHC practice).
How can I distinguish genuine EZH2 staining from a tissue artefact?
A credible EZH2 pattern is nuclear, with strong staining expected in tonsil germinal center cells and testis pachytene spermatocytes (UniProt Q15910: Nucleus; HPA: High in tonsil germinal center cells and testis pachytene spermatocytes). Treat predominantly cytoplasmic color or equally strong staining in unrelated compartments as a prompt to inspect specificity and detection controls (HPA: Nucleoplasm; standard IHC practice). Check whether intense color follows section edges, folds or necrotic areas, since those patterns can arise from processing or reagent accumulation (standard IHC practice). If signal persists in a no-primary control, investigate endogenous enzyme activity or chromogenic background before interpreting it as EZH2 expression (standard IHC practice).
Boster reagents
Best EZH2 / Histone-lysine N-methyltransferase EZH2 IHC Antibodies
Anti-EZH2 antibodies have IHC images from human tonsil and IF or ICC images from human cells (catalog image captions). IHC-listed options cover Human, Mouse and Rat (catalog reactivity).
Real IHC data
Anti-KMT6 / EZH2 Rabbit Monoclonal Antibody
Cat # M00050-1
Real IHC data
Anti-EZH2 Rabbit Monoclonal Antibody
Cat # M00050-2
Real IF data
Anti-EZH2 Antibody
Cat # A00050
Real IF data
Anti-EZH2 Antibody
Cat # A00050-3
M00050-1 has a paraffin-embedded human tonsil IHC image (catalog image caption); M00050-2 has a human tonsil IHC image with processing unreported (catalog image caption). A00050 has an ICC image in 293 cells (catalog image caption); A00050-3 has an IF image in paraformaldehyde-fixed HeLa cells (catalog image caption).
Which to pick: For paraffin-section IHC, start with M00050-1, a rabbit monoclonal with a paraffin-embedded tonsil image and an IHC dilution of 1:50–1:200; its fixative is unreported (catalog applications, dilution and image caption). For cell imaging, A00050 has a 293-cell ICC image, while A00050-3 has a HeLa IF image and is also listed for IHC-P (catalog image captions and applications). For Human, Mouse or Rat samples, M00050-1 and M00050-2 are both rabbit monoclonals listed for IHC and IF across those species; M00050-2’s tonsil caption does not report tissue processing or fixation (catalog reactivity, applications and image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q15910 (EZH2_HUMAN, Histone-lysine N-methyltransferase EZH2).
- Human Protein Atlas. EZH2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. EZH2 subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. EZH2 antibody validation summary (2 antibodies).
- Evaluation of Immunohistochemical Expression of Enhancer of Zeste Homolog 2 (EZH2) and Its Association With Clinicopathological Variables in Carcinoma Cervix. Cureus 2023 — PMC10148987.
- Immunohistochemical loss of enhancer of Zeste Homolog 2 (EZH2) protein expression correlates with EZH2 alterations and portends a worse outcome in myelodysplastic syndromes. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 2022 — PMC13105204.
- EZH2 protein expression associates with the early pathogenesis, tumor progression, and prognosis of non-small cell lung carcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research 2013 — PMC3890101.
- Prognostic impact of c-MYC and EZH2 expression in small cell and non-small cell lung carcinoma: a single-center retrospective study. Discover oncology 2026 — PMC13172210.
- PubMed PMID:8954776 — UniProt-cited evidence.
- PubMed PMID:9214638 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for EZH2 (Histone-lysine N-methyltransferase EZH2). Expected localisation Nuclear staining in several tissues (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: M00050-1 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Bone marrow (Hematopoietic cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human tonsil tissue; fixative not specified (datasheet M00050-1); 2. Fixation: Image fixative and duration unreported (datasheet M00050-1); 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 monoclonal (clone IDF-5) anti-EZH2, 1:50-1:200 (datasheet M00050-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Staining varies by cell type within tissues (HPA tissue IHC); Isoform / epitope: 5 isoforms; epitope coverage is unspecified (UniProt); Regulation: Overexpressed in several tumor types (UniProt). Evidence: UniProt Q15910, HPA tissue IHC · Enhanced, HPA ICC-IF · nucleoplasm., Datasheet M00050-1, PMC10148987, PMC13105204. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/EZH2/immunohistochemistry-ihc-guide.