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TOP2B / DNA topoisomerase 2-beta · IHC design guide
Design Immunohistochemistry for TOP2B
Plan TOP2B paraffin IHC around its widespread nuclear tissue staining (HPA tissue IHC). Start the IHC-validated antibody at 1:100–1:300 (datasheet: A02750), using bone marrow hematopoietic cells as a positive tissue control (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
TOP2B 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 tissue (HPA tissue IHC) | |
| Staining pattern | Widespread nuclear staining across tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A02750) | |
| Caveat | Adipocytes may stain weakly despite the broad tissue pattern (HPA tissue IHC) | |
| Regulation | No intensity regulator reported (UniProt) | |
| Isoform / epitope | Two isoforms, Beta-1 and Beta-2; check epitope coverage (UniProt) |
Section 1
Recommended TOP2B IHC & IF Protocols
The catalog antibody IHC-P protocol is accompanied by 2 published TOP2B protocols using paraffin sections (PMC13060179 methods; PMC13223395 methods).
| Sample | Paraffin-embedded human lung carcinoma tissue; fixative not specified (datasheet A02750) |
| Fixation | Image fixative and duration unreported (datasheet A02750); 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-TOP2B, 1:100 - 1:300 (datasheet A02750) |
| 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 | TOP2B-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 retrieval at 95–98 °C for 20 min (page retrieval). Citrate pH 6 was used for glioma sections (PMC13060179 methods).
Section 2
What Is the Expected TOP2B Staining Pattern?
TOP2B should stain nuclei across many cell types: HPA reports ubiquitous nuclear expression with supported IHC reliability and low tissue RNA specificity (HPA: tissue IHC). UniProt places TOP2B in the nucleoplasm and nucleolus and reports no transmembrane segment (UniProt Q02880: subcellular location; topology). Assess staining in the relevant cells rather than treating an entire tissue section as uniformly positive (HPA: tissue IHC).
| Clear nuclear staining in glandular or hematopoietic cells. | This fits reported high staining in appendix, cervix, duodenum and epididymis glandular cells, and bone marrow hematopoietic cells (HPA: tissue IHC). Judge whether the signal is centered on nuclei with visible tissue structure; intensity alone does not establish antibody specificity (general IHC practice). |
| Predominantly cytoplasmic or membranous staining with little nuclear signal. | This conflicts with the reported nuclear pattern and the absence of a transmembrane segment (HPA: tissue IHC; UniProt Q02880: topology). Consider nonspecific staining or a detection artefact before interpreting it as TOP2B. Recheck the staining pattern against a known-positive section (general IHC practice). |
| Strong signal confined to an unexpected cell population. | Compare the cells with the tissue-specific HPA entries before calling the pattern abnormal: TOP2B expression is broadly distributed, and HPA supplies no negative tissue list here (HPA: tissue IHC). If the signal remains discordant, investigate cross-reactivity or endogenous detection activity with appropriate controls (general IHC practice). |
| Diffuse color across nuclei, cytoplasm and extracellular spaces. | Diffuse background obscures the nuclear pattern reported for TOP2B (HPA: tissue IHC). Uneven reagent coverage, inadequate blocking or insufficient washing can produce background in chromogenic IHC; a no-primary control helps distinguish detection background from primary-antibody-associated signal (general IHC practice). |
| No nuclear staining in a section expected to be positive. | Absent signal in an HPA high-staining cell population warrants a technical check, but it does not establish that TOP2B is absent from the sample (HPA: tissue IHC; general IHC practice). Compare a known-positive section processed alongside it, then review retrieval, antibody dilution and detection steps (general IHC practice). |
💡Expected TOP2B appearanceA credible positive is predominantly nuclear staining in the relevant cells, with readily detectable signal in an HPA high-staining population; dominant cytoplasmic, membranous or diffuse staining is suspect (HPA: tissue IHC; UniProt Q02880: subcellular location).
| Cell and tissue context | HPA reports high staining in selected glandular, hematopoietic and glial populations, plus cells in the cerebellar molecular layer; adipocytes are listed as low (HPA: tissue IHC). Compare like cell populations when judging intensity, since these entries do not define a universal negative tissue (HPA: tissue IHC). |
| Antibody-specific validation | HPA lists IHC as Supported for HPA024120 and CAB004601; no IHC status is supplied for HPA050441 (HPA: antibody validation). These ratings support use of the reported patterns as references, but do not validate every catalog antibody or every staining condition (HPA: antibody validation; general IHC practice). |
| IF/ICC Q&A: where should fluorescence appear? | Mainly in the nucleoplasm; HPA calls this location enhanced in its ICC-IF record, while UniProt also lists the nucleolus (HPA: subcellular ICC-IF; UniProt Q02880: subcellular location). This localization answer does not specify an IF/ICC protocol. |
| Isoforms and epitope coverage | UniProt lists Beta-1 and Beta-2 isoforms, but the supplied record does not map an antibody epitope to either one (UniProt Q02880: isoforms). An unexpected pattern cannot be assigned to isoform recognition from these data alone; consult the chosen antibody's documented epitope before making that interpretation (general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| Nuclei are weak in an HPA high-staining cell population. | The result conflicts with the reported high staining, but the payload provides no TOP2B-specific retrieval or fixation sensitivity data (HPA: tissue IHC). | Run a known-positive section in the same batch. Check the selected antibody's documented IHC-P retrieval, dilution and detection settings, then review section quality and reagent performance (general IHC practice). |
| Only cytoplasm or cell borders stain. | The compartment conflicts with nuclear localization and the reported lack of a transmembrane segment (HPA: tissue IHC; UniProt Q02880: topology). | Compare with a known-positive section and a no-primary control. Reassess antibody specificity and detection background before scoring the signal as TOP2B (general IHC practice). |
| Background covers the whole section. | A widespread deposit prevents reliable assessment of the expected nuclear pattern (HPA: tissue IHC). Background can arise during staining or chromogenic detection (general IHC practice). | Inspect no-primary and reagent controls; check washing, blocking and detection conditions, then repeat with the catalog antibody's documented IHC-P settings (general IHC practice). |
| A surprising cell type stains strongly. | HPA describes ubiquitous nuclear expression but reports intensity by cell population; a surprising signal is not automatically cross-reactivity (HPA: tissue IHC). | Identify the stained cells and compare them with HPA's matching tissue entry. If the pattern remains discordant, check no-primary controls and, where available, an independently validated antibody (HPA: tissue IHC; general IHC practice). |
| Adipocytes show strong, widespread staining. | HPA lists adipocytes as low, so strong staining merits scrutiny; the low entry is not proof that every adipocyte must be unstained (HPA: tissue IHC). | Confirm whether the deposit is nuclear, compare nearby cell populations, and inspect a no-primary control for background or endogenous detection activity (HPA: tissue IHC; general IHC practice). |
| Two antibodies produce different patterns. | The supplied HPA statuses differ by antibody and application; they do not establish equivalent IHC performance for all reagents (HPA: antibody validation). | Compare each antibody's documented IHC-P conditions and epitope information, then judge which pattern agrees with the HPA nuclear reference in matched cells (HPA: tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing TOP2B staining
A02750 · IHCImmunohistochemistry analysis of paraffin-embedded human lung carcinoma tissue, using TOP2B Antibody. The picture on the right is blocked wi
A02750-1 · IHCImmunohistochemistry (IHC) analyzes of Topo IIβ (A32) pAb in paraffin-embedded human lung carcinoma tissue at 1:100.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000077097-TOP2B/tissue/cerebral+cortex#imid_1376804
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000077097-TOP2B/tissue/colon#imid_1376738
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000077097-TOP2B/tissue/liver#imid_1376739
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000077097-TOP2B/tissue/kidney#imid_1376732
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA024120; green = TOP2B, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA024120; green = TOP2B, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for TOP2B IHC & IF
🧪Run bone marrow first and expect nuclear staining in hematopoietic cells (HPA: High in bone marrow hematopoietic cells; UniProt Q02880: nuclear localization). HPA detects TOP2B in all 45 scored tissues, so use no-primary and isotype controls for the negative; cells on the positive slide with counterstained nuclei but no nuclear chromogen are background comparators, not validated TOP2B-negative cells (HPA: no negative tissue rows; standard IHC practice).
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: None in HPA: TOP2B is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show TOP2B in A-431, U-251MG, U2OS, U2OS, siRNA 1 (10x), U2OS, siRNA 1 (40x), U2OS, siRNA 2 (10x), U2OS, siRNA 2 (40x), U2OS, scrambled (10x), U2OS, scrambled (40x), with annotated localisation: Nucleoplasm (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only section and a nonimmune control matched to the primary antibody’s host species and clonality; use TOP2B knockout material if available or a cognate peptide block as a biological specificity control (standard IHC practice; A02750 tissue-IHC caption: peptide-blocked section). Quench endogenous peroxidase in bone marrow, where myeloid cells can otherwise produce chromogenic background (standard IHC practice).
⚠️Feasibility: The A02750 paraffin-section lung carcinoma caption does not state a fixative, and the supplied evidence reports no target-specific fixation window or fixation effect (A02750 tissue-IHC caption). Antigen-retrieval dependence is unreported; optimize retrieval for paraffin sections as standard IHC practice (supplied evidence; standard IHC practice). The supplied evidence does not establish frozen sections or IF/ICC as easier, while endogenous peroxidase in marrow myeloid cells is a practical chromogenic artefact to control (supplied evidence; standard IHC practice).
HPA tissue IHC evidence for TOP2B
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — 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 |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Caudate | Glial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Glial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: TOP2B is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced TOP2B IHC Tips
Troubleshoot TOP2B chromogenic IHC by assessing nuclear staining, section quality, and controls; IF/ICC considerations are included separately (UniProt Q02880 localisation; HPA tissue IHC).
How should I optimize retrieval when TOP2B nuclear staining is weak?
Start with Tris-EDTA pH 9.0 heat-induced epitope retrieval at 95–98 °C for 20 min (page retrieval rule: nuclear antigen). Keep section thickness, cooling, antibody incubation, and chromogen development consistent while comparing a weak slide with a positive control, so the retrieval change can be evaluated (standard IHC practice; HPA tissue IHC: ubiquitous nuclear expression). If nuclei remain weak, test a shorter or longer heating interval on adjacent sections and inspect morphology before considering another buffer as a fallback (standard IHC practice). Score signal in intact nuclei, since TOP2B is nuclear and mainly nucleoplasmic (UniProt Q02880 localisation; HPA subcellular).
Could fixation explain uneven TOP2B staining across paraffin sections?
Target-specific sensitivity of TOP2B to fixative type or fixation duration is unknown from the supplied evidence; the A02750 image identifies paraffin-embedded lung carcinoma but does not state its fixative (A02750 caption). Record the actual fixative, duration, tissue thickness, and processing history for each block before attributing differences to fixation (standard IHC practice). Compare similarly processed sections with the same retrieval, antibody incubation, and chromogen development, then check whether nuclear detail remains intact (standard IHC practice; UniProt Q02880 localisation). Overprocessing can reduce accessible epitopes or damage morphology in IHC generally, but that observation would require controlled comparison before assigning a TOP2B-specific cause (standard IHC practice).
What staining pattern should count as TOP2B positive in tissue IHC?
Count convincing TOP2B signal within intact nuclei, especially the nucleoplasm; UniProt also lists the nucleolus as a nuclear location (HPA subcellular: mainly nucleoplasmic; UniProt Q02880 localisation). HPA describes ubiquitous nuclear tissue expression, with high staining in appendix glandular cells and bone marrow hematopoietic cells, so use the local tissue architecture to identify the stained cell population (HPA tissue IHC). TOP2B has no transmembrane segment, so a membrane-only rim is discordant with the supplied localisation evidence (UniProt Q02880 topology and localisation). Compare chromogen with the counterstain at higher magnification and exclude pigment, folds, and poorly preserved nuclei before scoring (standard IHC practice).
Can this stain distinguish TOP2B isoforms or modification states?
Do not assign an IHC-positive nucleus to Beta-1 or Beta-2 without epitope mapping and isoform-specific validation; the record lists 2 TOP2B isoforms but supplies no isoform-specific antibody evidence (UniProt Q02880 isoforms; A02750 caption). TOP2B includes a Toprim domain at residues 476–593 and a catalytic domain at 736–1189, while the supplied record also lists acetylated and phosphorylated residues (UniProt Q02880 domains and modified residues). Those features can matter when selecting an epitope, but the caption does not identify the recognized sequence (UniProt Q02880; A02750 caption). Peptide blocking in the selected image supports competition by its synthesized peptide; it alone does not establish isoform or modification specificity (A02750 caption; standard IHC interpretation).
How should I assess TOP2B when adding an IF multiplex panel?
For the separate IF/ICC workflow, pair nuclear TOP2B with a validated marker identifying the cell population of interest, and use a nuclear counterstain to assess overlap (UniProt Q02880 localisation; standard IF practice). Choose fluorophores after checking tissue autofluorescence in an unstained control, favoring a spectral channel with a clear signal-to-background margin (standard IF practice). TOP2B has no transmembrane segment and is mainly nucleoplasmic, so permeabilisation must permit antibody access to the nuclear epitope; optimize detergent exposure against nuclear morphology (UniProt Q02880 topology; HPA subcellular; standard IF practice). The paraffin lung carcinoma caption supplies no IF fixation, permeabilisation, or fluorophore conditions for A02750 (A02750 caption).
How can I reduce diffuse brown staining without losing nuclear signal?
First compare a no-primary control with the test section to identify detection-system or tissue background, then inspect whether brown product actually overlaps intact nuclei (standard chromogenic IHC practice; UniProt Q02880 localisation). Block endogenous peroxidase before horseradish peroxidase detection, use an appropriate protein block, and titrate the primary antibody and DAB development if diffuse signal persists (standard chromogenic IHC practice). Keep washes and section handling consistent across controls, because uneven reagent coverage can produce patchy staining (standard IHC practice). The selected A02750 paraffin lung carcinoma image includes synthesized-peptide blocking, which is useful competition evidence for that image but does not replace background controls in each run (A02750 caption; standard IHC practice).
How should I score TOP2B staining across heterogeneous tissue regions? ⚠ ANSWER MARKED FOR VERIFICATION
Define viable regions and cell classes before scoring, then report nuclear percentage positive and an H-score using intensity categories 0–3 (standard IHC scoring practice; UniProt Q02880 localisation). For an H-score, multiply each intensity category by its percentage of scored nuclei and sum the products, giving a 0–300 scale (standard IHC scoring practice). Normalize counts to the number of evaluable nuclei in each named cell population, or report positive-cell density per mm² of viable tissue when spatial abundance is the question (standard IHC scoring practice). Apply one threshold and acquisition setting across sections, and document excluded necrotic or folded areas (standard IHC practice).
When is a strong TOP2B stain likely to be artefactual?
Treat strong membrane-only or extracellular brown deposits as suspect because TOP2B is a nuclear protein without a transmembrane segment (UniProt Q02880 localisation and topology). Check whether staining follows intact nuclei in the expected identified cell population; HPA reports widespread nuclear expression, so a positive cell type should be judged against its morphology and local controls (HPA tissue IHC; standard IHC interpretation). Signal concentrated at section edges, folds, or necrotic areas can reflect processing or reagent artefacts, while a positive no-primary control can indicate endogenous enzyme or detection background (standard chromogenic IHC practice). The A02750 peptide-blocked image supports peptide competition in paraffin lung carcinoma, but interpret each new specimen with its own controls (A02750 caption; standard IHC practice).
Boster reagents
Best TOP2B / DNA topoisomerase 2-beta IHC Antibodies
Two anti-TOP2B antibodies have IHC images from paraffin-embedded human lung carcinoma (each SKU’s image caption); A02750 also lists IF/ICC for human and mouse (catalog applications and reactivity), without an IF image (catalog images).
Real IHC data
Anti-TOP2B/Topoisomerase Ii Beta Antibody
Cat # A02750
Real IHC data
Anti-Topo II Beta (A32) TOP2B Antibody
Cat # A02750-1
A02750 will render with an IHC image of paraffin-embedded human lung carcinoma, including a peptide-blocked comparison; it lists human and mouse reactivity and IF/ICC applications (A02750 image caption; catalog). A02750-1 will render with an IHC image of paraffin-embedded human lung carcinoma at 1:100; it lists human, mouse and rat reactivity (A02750-1 image caption; catalog).
Which to pick: For tissue IHC, either SKU has its own paraffin-section image; choose A02750 if the peptide-blocked comparison is useful (each SKU’s image caption). For IF/ICC, choose A02750 because it lists those applications and is polyclonal, although no IF image is supplied (A02750 catalog applications, dilution record and images). For rat samples, A02750-1 is the listed option; its IHC caption supports paraffin sections, but reports no fixative (A02750-1 catalog reactivity and image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q02880 (TOP2B_HUMAN, DNA topoisomerase 2-beta).
- Human Protein Atlas. TOP2B tissue IHC expression (reliability: Supported).
- Human Protein Atlas. TOP2B subcellular location (ICC-IF): Mainly localized to the nucleoplasm..
- Human Protein Atlas. TOP2B antibody validation summary (3 antibodies).
- PDS5A and TOP2B cooperate for chromatin recruitment via CTCF. bioRxiv : the preprint server for biology 2026 — PMC13060179.
- Androgen-induced TOP2B-mediated double-strand breaks and prostate cancer gene rearrangements. Nature genetics 2010 — PMC3157086.
- Novel Mechanism of and Therapeutic Approach for Anthracycline-Induced Cardiotoxicity. Cancer research communications 2026 — PMC13223395.
- Top2b is involved in the formation of outer segment and synapse during late-stage photoreceptor differentiation by controlling key genes of photoreceptor transcriptional regulatory network. Journal of neuroscience research 2017 — PMC5561520.
- PubMed PMID:1333583 — UniProt-cited evidence.
- PubMed PMID:8383537 — UniProt-cited evidence.
- PubMed PMID:2556712 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for TOP2B (DNA topoisomerase 2-beta). Expected localisation Nuclear staining in tissue (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A02750 at 1:100 - 1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung carcinoma tissue; fixative not specified (datasheet A02750); 2. Fixation: Image fixative and duration unreported (datasheet A02750); 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-TOP2B, 1:100 - 1:300 (datasheet A02750); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A02750); Caveat: Adipocytes may stain weakly despite the broad tissue pattern (HPA tissue IHC); Isoform / epitope: Two isoforms, Beta-1 and Beta-2; check epitope coverage (UniProt); Regulation: No intensity regulator reported (UniProt). Evidence: UniProt Q02880, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the nucleoplasm., Datasheet A02750, PMC13060179, PMC13223395. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/TOP2B/immunohistochemistry-ihc-guide.