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TOP2A / DNA topoisomerase 2-alpha · IHC design guide
Design Immunohistochemistry for TOP2A
Plan paraffin-section TOP2A IHC around the nuclear staining seen mainly in highly proliferative cells (HPA tissue IHC). This guide covers fixation consistency, controls, chromogenic detection and interpretation of staining across cell populations.
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 proliferative cells (HPA tissue IHC) | |
| Staining pattern | Nuclear signal mainly in highly proliferative 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) | |
| Caveat | Low-proliferation areas may show little staining (HPA tissue IHC) | |
| Regulation | Enriched in proliferative cells (HPA tissue IHC) | |
| Isoform / epitope | 4 isoforms; check whether the epitope is shared (UniProt) |
Section 1
Recommended TOP2A IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet) with four published TOP2A IHC methods (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 (page antigen retrieval); use the paper’s stated retrieval when reproducing its method.
Section 2
What Is the Expected TOP2A Staining Pattern?
TOP2A staining is mainly nuclear in highly proliferative cells (HPA: Enhanced IHC; nuclear profile). Tonsil germinal-center cells and bone-marrow hematopoietic cells stain strongly (HPA: High). TOP2A has no transmembrane segment (UniProt P11388 topology).
| Strong nuclear stain in tonsil germinal-center cells (HPA: High). | Expected pattern in proliferative cells (HPA: nuclear profile). |
| Predominantly membranous stain. | Unexpected compartment; assess nonspecific staining (UniProt P11388: no transmembrane segment). |
| Strong adipocyte stain. | Unexpected; assess cross-reactivity or endogenous activity (HPA: adipocytes not detected). |
| Diffuse stain across the section. | Possible background; check blocking and washes (standard IHC practice). |
| No nuclear stain in tonsil germinal centers. | Unexpected in a known-positive site; check assay controls (HPA: High). |
💡Expected TOP2A appearanceStrong nuclear stain in tonsil germinal-center cells (HPA: High); diffuse cytoplasmic stain is suspect (HPA: nuclear profile).
| Cell context | Nuclear staining favors highly proliferative cells (HPA: tissue IHC profile). |
| IF/ICC: where is signal? | Mainly nucleoplasm; also nucleoli (HPA: supported ICC-IF localization). |
| IHC antibody validation | 3 listed antibodies have Enhanced IHC validation (HPA: antibodies). |
| Isoforms | 4 isoforms are listed; antibody epitope coverage is unreported (UniProt P11388). |
| Situation | Likely cause | Next action |
|---|---|---|
| No nuclear stain in tonsil germinal centers. | Signal is expected there (HPA: High); assay failure is possible. | Check positive control, retrieval and detection (standard IHC practice). |
| Diffuse background. | Blocking or washing may be inadequate (standard IHC practice). | Optimize blocking and washes; run a no-primary control (standard IHC practice). |
| Membranous stain dominates. | Unexpected for TOP2A (UniProt P11388: no transmembrane segment). | Check controls and antibody specificity (standard IHC practice). |
| Strong adipocyte stain. | Unexpected cell type (HPA: adipocytes not detected); cross-reactivity is possible. | Compare with a no-primary control and an IHC-validated antibody (HPA: Enhanced IHC). |
| Only some tonsil cells stain. | Signal favors proliferative germinal-center cells (HPA: nuclear profile; High). | Assess germinal-center nuclei separately (HPA: tonsil germinal-center cells High). |
| Stain persists without primary antibody. | Endogenous detection activity is possible (standard IHC practice). | Check the detection control and block endogenous activity (standard IHC practice). |
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 strong TOP2A staining (HPA: High in germinal center cells). Use adipose tissue as the negative, where adipocytes are not detected (HPA: Not detected in adipocytes); on the lymph node slide, cells verified as internal negatives should show little or no specific nuclear chromogen (HPA: High in germinal center cells; UniProt P11388: nuclear localization).
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 a no-primary (secondary-only) control, a concentration-matched isotype control matching the primary antibody’s host species and clonality, and a TOP2A knockout sample or peptide-block control if available (standard IHC practice). Block endogenous peroxidase and check for background staining in lymph node, particularly in myeloid cells (standard chromogenic IHC practice).
⚠️Feasibility: The supplied evidence reports no TOP2A-specific fixation window or retrieval dependence; optimize heat-induced antigen retrieval empirically for paraffin IHC (supplied target/application evidence; standard IHC practice). The M00953-1 paraffin-section caption does not report a fixative, and the supplied evidence does not establish that frozen sections or IF/ICC are easier (selected-SKU caption; HPA: ICC-IF images). Lymph node myeloid cells can contribute endogenous peroxidase background, so assess it with the no-primary control (standard chromogenic 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 in paraffin sections by checking retrieval, nuclear localisation, controls, and scoring before interpreting differences between samples.
How should I retrieve TOP2A in paraffin sections when nuclear staining is weak?
For TOP2A paraffin IHC, use Tris-EDTA pH 9.0 heat retrieval at 95–98 °C for 20 min (page retrieval specification). Compare sections cooled in retrieval buffer for the same interval, then process them with identical antibody incubation and chromogenic detection conditions so retrieval is the main variable (standard IHC practice). If staining remains weak, check whether expected germinal center cells stain before extending retrieval time in a small pilot series (HPA tissue IHC; standard IHC practice). Record tissue detachment, damaged morphology, and nuclear background alongside signal intensity, because these observations help identify excessive retrieval (standard IHC practice).
Could fixation explain weak or uneven TOP2A staining?
Target-specific fixation sensitivity for TOP2A is unknown from the supplied evidence, and the selected paraffin-section caption does not report a fixative (selected M00953-1 caption). Record the actual fixative, time before fixation, and fixation duration for each specimen before comparing staining intensity (standard IHC practice). For a controlled pilot, process matched pieces using a documented 10% neutral buffered formalin workflow and vary only one handling step at a time (standard IHC practice). Keep section thickness, pH 9.0 retrieval, and detection conditions constant; differences then support a handling effect without establishing a TOP2A-specific fixation mechanism (page retrieval specification; standard IHC practice).
What should I do if TOP2A staining looks cytoplasmic instead of nuclear?
First inspect the hematoxylin counterstain and score chromogen within intact nuclei separately from diffuse cytoplasmic colour (standard IHC practice). Predominantly nuclear staining in highly proliferative cells is the tissue-IHC pattern, while nucleoplasmic and additional nucleolar localisation are supported by subcellular imaging (HPA tissue IHC; HPA subcellular). UniProt also lists cytoplasmic localisation, so a cytoplasmic signal alone cannot establish either specificity or artefact without appropriate controls (UniProt P11388 subcellular). Compare a stained section with its no-primary control and review germinal center cells as an expected positive population before accepting an unusual compartment pattern (HPA tissue IHC; standard IHC practice).
Could isoform choice or epitope masking change my TOP2A IHC result?
TOP2A has 4 listed isoforms, but the supplied product caption does not identify the antibody epitope or its isoform coverage (UniProt P11388 isoforms; selected M00953-1 caption). The protein contains a Toprim domain at residues 455–572 and a catalytic domain at 715–1171, with multiple modified residues (UniProt P11388 domains and modified residues). Those features justify checking an available epitope map, but they do not establish that a particular modification or isoform changes staining (UniProt P11388; standard IHC practice). If two antibodies disagree, compare their documented epitopes, retrieval conditions, and nuclear staining in the same positive tissue before assigning the difference to biology (HPA tissue IHC; standard IHC practice).
How can I assess TOP2A by multiplex IF alongside this IHC guide?
Treat IF as a separate assay and pilot the antibody with a nuclear counterstain plus a marker identifying the expected cell population, such as CD20 for germinal center B cells (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores and place the weaker signal in a far-red channel if tissue autofluorescence is prominent, checking single-stain controls for bleed-through (standard IF practice). TOP2A has no transmembrane segment and is mainly nucleoplasmic, so use a mild permeabilisation pilot to access intracellular epitopes rather than assuming a membrane-facing epitope (UniProt P11388 topology; HPA subcellular; standard IF practice). Compare stained and no-primary controls with matched exposures, and do not transfer the paraffin-IHC caption into an IF fixation claim (selected M00953-1 caption; standard IF practice).
How do I reduce diffuse brown background without losing TOP2A-positive nuclei?
Check a no-primary section first to separate detection-system colour from antibody-dependent staining, and inspect whether background follows tissue edges or damaged regions (standard IHC practice). For peroxidase-based chromogenic IHC, a 3% hydrogen peroxide block for about 10 min is a general workflow starting point, not TOP2A-specific evidence (standard IHC practice). Titrate the primary antibody and blocking conditions on matched sections while keeping pH 9.0 retrieval constant, then compare nuclear signal with nonnuclear colour (page retrieval specification; standard IHC practice). Germinal center cells provide an expected positive population, whereas adipocytes are reported as undetected and can help flag widespread staining (HPA tissue IHC).
What is a defensible way to quantify TOP2A-positive cells in IHC? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue region and cell population before scoring, then count only interpretable nuclei within viable areas (standard IHC practice). Report the percentage of positive nuclei with the number of nuclei assessed; if intensity matters, use an H-score from 0–300 based on percentages at intensity grades 0–3 (standard IHC practice). For spatial comparisons, report positive nuclei per mm² and normalise cell-level results to the number of eligible nuclei in the same region (standard IHC practice). Keep thresholds and imaging conditions fixed across specimens, because TOP2A expression is concentrated in highly proliferative cells and cell composition can shift the overall score (HPA tissue IHC; standard IHC practice).
How can I distinguish a genuine TOP2A-positive nucleus from artefact?
A convincing result places chromogen in intact nuclei of an expected proliferative population, such as lymph-node germinal center cells (HPA tissue IHC; standard IHC practice). Compare the pattern with a no-primary control and examine the counterstain before interpreting diffuse cytoplasmic colour or signal in an unexpected cell population (standard IHC practice). Discount staining confined to cut edges, necrotic regions, or endogenous peroxidase-rich areas until section quality and detection controls have been checked (standard IHC practice). Interpret focal absence cautiously: TOP2A tissue staining is mainly seen in highly proliferative cells, and the selected paraffin-section caption alone does not establish diagnostic specificity (HPA tissue IHC; selected M00953-1 caption).
Boster reagents
Best TOP2A / DNA topoisomerase 2-alpha IHC Antibodies
The catalog includes TOP2A antibodies with IHC images from human paraffin sections and an IF/ICC image from U20S cells (catalog image captions). Both list human, mouse and rat reactivity (catalog reactivity).
Real IHC data
Anti-Topoisomerase II alpha TOP2A Rabbit Monoclonal Antibody
Cat # M00953-1
The rendered card is M00953-1, with an IHC image from paraffin-embedded human colon cancer (M00953-1 IHC image caption). M00953-1 lists IHC, IP and WB applications and human, mouse and rat reactivity (M00953-1 catalog applications and reactivity).
Which to pick: For tissue IHC, choose M00953-1: it is a rabbit monoclonal with an image from a human paraffin section; the caption does not report the fixative (M00953-1 catalog clone and IHC image caption). For IF/ICC, choose A00953, which lists both applications and has an IF/ICC image from U20S cells (A00953 catalog applications and IF image caption). For mouse or rat tissue IHC, both SKUs list reactivity with those species, while their IHC image captions show human paraffin sections (A00953 and M00953-1 catalog reactivity and IHC image 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 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); Caveat: Low-proliferation areas may show little staining (HPA tissue IHC); Isoform / epitope: 4 isoforms; check whether the epitope is shared (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.