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- Table of Contents
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.
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
| 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 |
| 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) |
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. |
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. |
| 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. |
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.
| 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 → |
| 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 → |
Troubleshoot EZH2 staining by checking nuclear localisation, cell type, tissue processing and controls before comparing signal across sections.
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).
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).