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TOP2A / DNA topoisomerase 2-alpha · IHC design guide
Design Immunohistochemistry for TOP2A
Plan chromogenic TOP2A IHC on paraffin sections using germinal center cells as a positive reference (HPA tissue IHC). This guide covers nuclear scoring and the catalog antibody’s 2–5 μg/mL IHC dilution (datasheet).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
TOP2A 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 highly proliferative cells (HPA tissue IHC) | |
| Staining pattern | Nuclear staining in 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 sections (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M00953-1) | |
| Caveat | Staining varies with proliferative cell fraction (HPA tissue IHC) | |
| Regulation | Enriched in proliferative cells (HPA tissue IHC) | |
| Isoform / epitope | Four isoforms; epitope differences are unspecified (UniProt) |
Section 1
Recommended TOP2A IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by four published TOP2A paraffin-section workflows (PMC10011289; PMC2613415; PMC3140260; PMC8761301).
| Sample | Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet M00953-1) |
| Fixation | Image fixative and duration unreported (datasheet M00953-1); 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 FB-20) anti-TOP2A, 1:50-1:200 (datasheet M00953-1) |
| 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 | TOP2A-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: Nuclear expression mainly observed in highly proliferative cells. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 at 95–98 °C for 20 min for the catalog antibody (page antigen-retrieval specification).
Section 2
What Is the Expected TOP2A Staining Pattern?
TOP2A should stain nuclei chiefly in highly proliferative cells, including germinal center cells and hematopoietic cells (HPA: tissue IHC). The tissue pattern has Enhanced reliability, with high consistency between antibody staining and RNA expression (HPA: tissue IHC). UniProt lists nuclear and cytoplasmic locations and no transmembrane segment (UniProt P11388: subcellular location; topology).
| Distinct nuclear staining in germinal centers or bone marrow hematopoietic cells. | This matches the reported high staining in those cells and the predominantly nuclear pattern in highly proliferative cells (HPA: tissue IHC). Compare positive nuclei with adjacent cells on the same section; a positive tissue can contain cells with different staining levels (HPA: tissue IHC). |
| Predominantly cytoplasmic staining, a membrane rim, or nuclear staining without a discernible cell pattern. | Predominantly nuclear tissue staining is expected (HPA: tissue IHC). UniProt also lists cytoplasm, so cytoplasmic signal alone cannot establish an artefact (UniProt P11388: subcellular location). Check whether the signal follows cell boundaries or persists in a detection-only control (general IHC practice). |
| Strong staining in adipocytes or cardiomyocytes. | Those cell types are reported as not detected (HPA: adipose tissue; heart muscle). Review morphology and compare with a reagent control before assigning the signal to TOP2A; cross-reactivity or endogenous detection activity is possible (general IHC practice). Do not extend a cell-specific negative result to every cell in that tissue. |
| Diffuse brown signal across tissue, including extracellular areas or cells expected to be negative. | This does not resemble the nuclear, proliferation-associated tissue pattern (HPA: tissue IHC). Uneven chromogen deposition, nonspecific antibody binding, or endogenous enzyme activity can produce broad background (general IHC practice). Assess the no-primary control and tissue edges before scoring nuclei. |
| No convincing nuclei stain in a known-positive germinal center or bone marrow section. | High staining is reported for germinal center cells and bone marrow hematopoietic cells (HPA: tissue IHC). A blank positive control makes the run inconclusive; examine tissue identity, retrieval, antibody application, detection reagents, and counterstain visibility (general IHC practice). It does not establish absent TOP2A expression in the test specimen. |
💡Expected TOP2A appearanceCall a result positive when discrete nuclear chromogen marks proliferative cells, with high staining possible in germinal centers or bone marrow hematopoietic cells (HPA: tissue IHC); diffuse extracellular or predominantly membrane-associated color is suspect (HPA: nuclear tissue pattern; general IHC practice).
| Cell population and section context | The expected signal is cell selective: germinal center cells, marrow hematopoietic cells, cytotrophoblasts, and pachytene spermatocytes are high; basal skin cells are medium (HPA: tissue IHC). Score identified cells rather than calling an entire organ uniformly positive. |
| Compartment and IF/ICC comparison | IF/ICC? HPA supports nucleoplasm as the main location and nucleoli as an additional location in cell images (HPA: subcellular ICC-IF). Use that as a location cross-check, while assessing chromogenic tissue sections against HPA’s predominantly nuclear IHC pattern (HPA: tissue IHC). |
| Antibody validation | Three listed antibodies have Enhanced IHC validation (HPA: antibodies HPA006458, HPA026773, CAB002448). This supports the reported pattern across validated reagents; it does not establish that an unlisted catalog antibody recognizes the same epitope or performs identically in the current section (HPA: antibody validation scope). |
| Molecular variants and epitope uncertainty | UniProt lists four isoforms and multiple modified residues, including phosphorylation sites (UniProt P11388: isoforms; modified residues). The payload gives no antibody epitope or isoform coverage, so staining cannot be assigned to a particular isoform or modification from this pattern alone. |
| Retrieval and detection controls | Antigen retrieval and detection conditions are general IHC workflow variables (general IHC practice). No supplied source reports TOP2A-specific fixation or retrieval sensitivity; use matched controls to judge the run without treating a retrieval response as a known property of TOP2A. |
| Situation | Likely cause | Next action |
|---|---|---|
| Positive-control tissue is blank. | A missed primary-antibody step, ineffective detection, or unsuitable retrieval may prevent visible staining (general IHC practice). | Check the control’s germinal center or marrow cells, which are reported high (HPA: tissue IHC); verify reagent application and detection, then repeat with a documented IHC workflow (general IHC practice). |
| Test tissue is negative, but the positive control stains. | The sampled cells may have little detectable protein; HPA reports cell-dependent staining, including not-detected populations (HPA: tissue IHC). | Identify the relevant cells before interpreting the negative result; compare cell morphology and a suitable positive reference on the same run (general IHC practice). Avoid calling an entire tissue negative from one cell population. |
| Most cells show brown cytoplasm with weak or absent nuclear definition. | The pattern differs from predominantly nuclear tissue staining (HPA: tissue IHC); nonspecific binding or detection background is possible (general IHC practice). | Inspect a no-primary control, reduce excessive chromogen development if present, and reassess nuclear boundaries (general IHC practice). UniProt also lists cytoplasm, so retain that location caveat (UniProt P11388: subcellular location). |
| Adipocytes or cardiomyocytes stain strongly. | Those cell types are reported not detected (HPA: adipose tissue; heart muscle); cross-reactivity or endogenous detection activity may explain unexpected color (general IHC practice). | Confirm cell identity, review a no-primary control, and use an independently validated IHC antibody if available (general IHC practice; HPA: antibody validation). Do not infer that every cell in either tissue must be negative. |
| Background obscures nuclei across the section. | Nonspecific binding, endogenous enzyme activity, or overdeveloped chromogen can obscure cell-localized signal (general IHC practice). | Compare the no-primary control, check blocking and detection conditions, and shorten chromogen development when excessive (general IHC practice). Score only nuclei that remain distinguishable within the expected cell populations (HPA: tissue IHC). |
| Nucleolar detail in IF/ICC seems inconsistent with the IHC slide. | HPA’s ICC-IF summary includes nucleoli alongside the main nucleoplasmic location (HPA: subcellular ICC-IF), while tissue IHC is summarized as nuclear staining (HPA: tissue IHC). | Interpret each image using its application’s reported resolution: use nuclear cell distribution for chromogenic IHC and nucleoplasm or nucleoli for IF/ICC location review (HPA: tissue IHC; subcellular ICC-IF). |
Experiment evidence
IHC & IF images showing TOP2A staining
M00953-1 · IHCImmunohistochemical analysis of paraffin-embedded human colon cancer, using Topoisomerase II alpha Antibody.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000131747-TOP2A/tissue/cerebral+cortex#imid_755336
HPA · EsophagusHuman Protein Atlas IHC, Esophagus. Source: https://www.proteinatlas.org/ENSG00000131747-TOP2A/tissue/esophagus#imid_755308
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000131747-TOP2A/tissue/colon#imid_755254
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000131747-TOP2A/tissue/liver#imid_755263
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB002448; green = TOP2A, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB002448; green = TOP2A, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for TOP2A IHC & IF
🧪Run lymph node first: germinal center cells should show TOP2A staining (HPA: High in germinal center cells). Use adipose tissue as the negative tissue (HPA: Not detected in adipocytes); on the positive slide, unstained nuclei outside the stained germinal center population can serve as internal negatives, but confirm their staining status on the slide.
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 TOP2A in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and isotype controls matched to the primary antibody’s host species and clonality, plus a TOP2A knockout biological negative where available (standard IHC practice). For chromogenic detection, quench endogenous peroxidase and block endogenous biotin if using avidin–biotin detection (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and retrieval dependence are unreported in the supplied evidence; optimize antigen retrieval empirically for paraffin IHC (standard IHC practice). The M00953-1 tissue-IHC caption describes a paraffin-embedded specimen but leaves the fixative unreported (selected-SKU caption). The supplied evidence does not establish whether frozen sections or IF are easier; in lymph node, dense germinal center nuclei can complicate cell-by-cell scoring (standard IHC practice).
HPA tissue IHC evidence for TOP2A
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 → |
| Lymph node | Germinal center cells | High | Protein (IHC) | HPA → |
| Placenta | Cytotrophoblasts | High | Protein (IHC) | HPA → |
| Skin | Cells in basal layer | Medium | Protein (IHC) | HPA → |
| Testis | Pachytene spermatocytes | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | 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 → |
| Epididymis | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced TOP2A IHC Tips
Troubleshoot TOP2A staining by checking retrieval, nuclear localisation and the distribution of positive cells in viable tissue (page retrieval rule: nuclear antigen; HPA: tissue IHC profile).
How should I troubleshoot weak TOP2A staining after antigen retrieval?
Use Tris-EDTA at pH 9.0 for heat-induced epitope retrieval at 95–98 °C for 20 min (page retrieval rule: nuclear antigen). If nuclear staining is weak, confirm the buffer pH, heating time and full immersion of each paraffin section before changing conditions (general IHC practice). Run a known positive tissue alongside the test section; tonsil germinal center cells show high TOP2A expression and provide a useful reference (HPA: tissue IHC). If staining is uneven or tissue lifts, adjust section adhesion and cooling conditions, then compare nuclear signal and morphology across sections (general IHC practice).
Could fixation explain weak or inconsistent TOP2A staining in paraffin sections?
The selected antibody image documents staining in paraffin-embedded human colon cancer, but its caption does not state the fixative (catalog antibody M00953-1: tissue IHC caption). Target-specific sensitivity to fixation is therefore unknown from the supplied evidence (catalog antibody M00953-1: tissue IHC caption). Record the fixative, fixation duration, processing history and section age for each case before attributing a weak result to fixation (general IHC practice). Compare sections processed under known, consistent conditions and assess nuclear staining in an appropriate positive control; avoid changing fixation and retrieval conditions simultaneously (general IHC practice; HPA: nuclear expression in highly proliferative cells).
What localisation should count as convincing TOP2A staining?
Score crisp staining within viable cell nuclei as the primary pattern in chromogenic sections (HPA: tissue IHC profile shows nuclear expression mainly in highly proliferative cells). TOP2A is mainly nucleoplasmic, with additional nucleolar localisation reported in cell imaging (HPA: subcellular localisation). UniProt also lists cytoplasmic localisation, so a cytoplasmic signal alone should prompt comparison with nuclear staining and controls rather than automatic acceptance as specific (UniProt P11388: subcellular localisation; general IHC practice). Inspect the corresponding counterstain for intact nuclei and distinguish discrete nuclear signal from diffuse pigment, tissue folds or staining concentrated at section edges (general IHC practice).
Could isoforms or epitope masking change the apparent TOP2A pattern?
TOP2A has 4 listed isoforms, but the supplied catalog caption does not identify the antibody epitope or establish which isoforms it detects (UniProt P11388: isoforms; catalog antibody M00953-1: tissue IHC caption). The protein contains a Toprim region at residues 455–572 and a catalytic region at 715–1171; these positions do not locate the antibody epitope (UniProt P11388: domains; catalog antibody M00953-1: tissue IHC caption). Multiple modified residues are reported, including phosphorylation, but an effect on this antibody's staining is unestablished (UniProt P11388: modified residues). If results conflict, obtain epitope documentation and compare a separately validated antibody with a mapped, distinct epitope under matched IHC conditions (general IHC practice).
How can I cross-check TOP2A staining with multiplex immunofluorescence?
Use IF as a separate cross-check, since the selected antibody evidence supplied here is a paraffin-section IHC caption rather than an IF validation (catalog antibody M00953-1: tissue IHC caption). Pair TOP2A with a validated marker for the expected cell population, such as germinal center cells in tonsil, and assess whether nuclear signals occur in those cells (HPA: high expression in tonsil germinal center cells; general IF practice). Choose a fluorophore channel after examining tissue autofluorescence and spectral overlap, with a longer-wavelength channel if background is stronger at shorter wavelengths (general IF practice). Because TOP2A is nucleoplasmic and has no transmembrane segment, optimise permeabilisation for access across cellular and nuclear membranes while preserving nuclear morphology (HPA: subcellular localisation; UniProt P11388: topology; general IF practice).
How do I reduce diffuse or widespread chromogenic TOP2A background?
Check a no-primary control to identify signal from the detection system, then review blocking, wash conditions and primary-antibody concentration against the matched positive section (general IHC practice). In a peroxidase and DAB workflow, apply an endogenous peroxidase block and examine whether residual colour persists without primary antibody (general IHC practice). Avoid calling uniform staining across unrelated cell populations positive when the expected tissue pattern is predominantly nuclear in highly proliferative cells (HPA: tissue IHC profile). Compare a high-expression reference, such as bone marrow hematopoietic cells, with a low-signal reference where appropriate, and interpret both with intact morphology (HPA: tissue IHC; general IHC practice).
What should I count when comparing TOP2A IHC across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define viable regions before scoring and count positive nuclei within a specified cell population, because the reported tissue pattern is predominantly nuclear and linked to highly proliferative cells (HPA: tissue IHC profile; general IHC practice). Report the percentage of positive nuclei with the total eligible nuclei as its denominator, using a consistent staining threshold across slides (general IHC practice). If intensity is reproducible, an H-score sums the percentage at each 0–3 intensity level multiplied by that level, yielding 0–300 (general IHC scoring practice). For spatial comparisons, report positive nuclei per mm² of viable tissue and document excluded necrotic or folded areas (general IHC practice).
How can I distinguish genuine TOP2A positivity from artefact?
Look for defined nuclear staining in viable, morphologically identifiable cells, particularly in proliferative compartments, rather than colour alone (HPA: tissue IHC profile; general IHC practice). Strong diffuse cytoplasmic staining without a corresponding nuclear pattern warrants review of background and detection controls, although cytoplasmic localisation is also listed for TOP2A (UniProt P11388: subcellular localisation; general IHC practice). Exclude section-edge concentration, folds and necrotic areas when judging distribution or scoring positive cells (general IHC practice). If pigment persists in a no-primary control, investigate endogenous peroxidase or other detection-related background before assigning it to TOP2A (general IHC practice).
Boster reagents
Best TOP2A / DNA topoisomerase 2-alpha IHC Antibodies
Catalog anti-TOP2A antibodies show human paraffin-section IHC in colon, lung and rectal cancer (M00953-1 and A00953 IHC captions), plus IF in U20S cells (A00953 IF caption).
Real IHC data
Anti-Topoisomerase II alpha TOP2A Rabbit Monoclonal Antibody
Cat # M00953-1
M00953-1 is the rendered card, with IHC shown in paraffin-embedded human colon cancer (M00953-1 IHC caption). Its catalog lists human, mouse and rat reactivity; the image documents human tissue (M00953-1 catalog reactivity; M00953-1 IHC caption).
Which to pick: For tissue IHC, choose M00953-1, a rabbit monoclonal antibody, clone FB-20, with an IHC dilution of 1:50–1:200 and a paraffin-section image from human colon cancer; the fixative is unreported (M00953-1 catalog; M00953-1 IHC caption). For IF/ICC, choose A00953, which lists both applications and has an IF image in U20S cells (A00953 catalog applications; A00953 IF caption). For work across species, both list human, mouse and rat reactivity, while their IHC captions document human tissues only (M00953-1 and A00953 catalog reactivity and IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P11388 (TOP2A_HUMAN, DNA topoisomerase 2-alpha).
- Human Protein Atlas. TOP2A tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. TOP2A subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the nucleoli..
- Human Protein Atlas. TOP2A antibody validation summary (3 antibodies).
- TOP2A Expression in Pheochromocytoma and Abdominal Paraganglioma: a Marker of Poor Clinical Outcome?. Endocrine pathology 2023 — PMC10011289.
- Analysis of EGFR, HER2, and TOP2A gene status and chromosomal polysomy in gastric adenocarcinoma from Chinese patients. BMC cancer 2008 — PMC2613415.
- Novel molecular markers of malignancy in histologically normal and benign breast. Pathology research international 2011 — PMC3140260.
- Study on the expression of TOP2A in hepatocellular carcinoma and its relationship with patient prognosis. Cancer cell international 2022 — PMC8761301.
- PubMed PMID:2845399 — UniProt-cited evidence.
- PubMed PMID:8393377 — UniProt-cited evidence.
- PubMed PMID:9795238 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for TOP2A (DNA topoisomerase 2-alpha). Expected localisation Nuclear staining in highly proliferative cells (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: M00953-1 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 colon cancer tissue; fixative not specified (datasheet M00953-1); 2. Fixation: Image fixative and duration unreported (datasheet M00953-1); 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 FB-20) anti-TOP2A, 1:50-1:200 (datasheet M00953-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M00953-1); Caveat: Staining varies with proliferative cell fraction (HPA tissue IHC); Isoform / epitope: Four isoforms; epitope differences are unspecified (UniProt); Regulation: Enriched in proliferative cells (HPA tissue IHC). Evidence: UniProt P11388, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet M00953-1, PMC10011289, PMC2613415. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/TOP2A/immunohistochemistry-ihc-guide.