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CCNB2 / G2/mitotic-specific cyclin-B2 · IHC design guide
Design Immunohistochemistry for CCNB2
Plan CCNB2 IHC on paraffin sections using the catalog antibody at 1:50 (datasheet M02040). Assess cytoplasmic staining in a subset of cells (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CCNB2 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 | Cytoplasmic staining in a subset of cells (HPA tissue IHC) | |
| Staining pattern | A subset of cells shows cytoplasmic staining across tissues (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M02040) | |
| Positive control | Appendix+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). Selected-image fixative and duration unreported (datasheet M02040); verify before use. | |
| Caveat | The G2/M cell-cycle role may limit the positive-cell fraction (UniProt) | |
| Regulation | G2/mitotic-specific expression (UniProt) | |
| Isoform / epitope | 0 annotated isoforms; one unprocessed chain (UniProt) |
Section 1
Recommended CCNB2 IHC & IF Protocols
The catalog antibody protocol is accompanied by 4 published CCNB2 IHC protocols covering breast and gastric cancer tissue (PMC8728388; PMC11832750; PMC3545739; PMC8331305).
| Sample | Paraffin-embedded human colon adenocarcinoma tissue; fixative not specified (datasheet M02040) |
| Fixation | Image fixative and duration unreported (datasheet M02040); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet M02040); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M02040) |
| Primary antibody | Rabbit monoclonal (clone GIG-3) anti-CCNB2, 1:50 (datasheet M02040) |
| Primary incubation | Overnight at 4 °C (datasheet M02040) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet M02040) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CCNB2-positive staining in endocrine cells of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in a subset of cells in several tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet M02040); a breast carcinoma study used EDTA at pH 9 (PMC3545739).
Section 2
What Is the Expected CCNB2 Staining Pattern?
Expect CCNB2 staining in the cytoplasm of a subset of cells, including endocrine cells in appendix and colon, hematopoietic cells in bone marrow, and Leydig cells in testis (HPA tissue IHC: High in each listed cell type). HPA rates the tissue staining Enhanced, with medium consistency between antibody staining and RNA data (HPA tissue IHC). CCNB2 has no transmembrane segment (UniProt O95067 topology).
| Distinct cytoplasmic staining in a subset of the expected cells, with unstained neighbors. | This matches the reported tissue pattern. Strong staining in the listed positive cell types is plausible; the HPA profile does not require every cell in a section to stain (HPA tissue IHC: cytoplasmic expression in a subset; High in listed cells). |
| Predominantly nuclear or membrane outlined staining in the expected cells. | Recheck the pattern before calling it CCNB2. HPA reports cytoplasmic tissue staining, while UniProt lists no transmembrane segment (HPA tissue IHC; UniProt O95067 topology). A mismatched compartment may reflect staining artefact; localisation alone cannot identify its cause (general IHC practice). |
| Strong staining in adipocytes, cardiomyocytes, or lung alveolar cells. | These cells are reported as not detected, so strong staining warrants a specificity check (HPA tissue IHC: Not detected in each listed cell type). Cross reactivity or endogenous detection activity are possible explanations; the pattern alone cannot distinguish them (general IHC practice). |
| Broad, hazy chromogen across cells and surrounding tissue. | Diffuse background obscures the subset pattern reported by HPA (HPA tissue IHC). Examine a no primary control and background outside cells before scoring; nonspecific reagent binding or endogenous detection activity can produce background (general IHC practice). |
| No signal in bone marrow hematopoietic cells or testis Leydig cells. | Treat a blank positive control as an assay problem to investigate before interpreting study sections (HPA tissue IHC: High in both cell types). Check cell identity, antibody and detection performance, and section handling (general IHC practice). |
💡Expected CCNB2 appearanceCall a result consistent with the reference pattern when cytoplasmic staining in a subset includes strong signal in an HPA High cell type; broad haze or strong signal in HPA Not detected cell types is suspect (HPA tissue IHC; general IHC practice).
| Cell selection and scoring | Score the named cell population, not the whole organ: HPA reports High staining in selected endocrine, hematopoietic, and Leydig cells, alongside a cytoplasmic subset pattern (HPA tissue IHC). A mostly unstained field can therefore contain a valid positive subset. |
| Cell cycle context | CCNB2 acts at the G2/M transition, so cell cycle state may help interpret variation among cells (UniProt O95067 function). HPA tissue staining alone does not establish that every stained cell is mitotic or define a staining threshold for cell cycle phase (HPA tissue IHC). |
| Antibody evidence | Two listed antibodies have Enhanced IHC validation, and HPA rates the tissue profile Enhanced (HPA antibodies: HPA008873 and CAB009575; HPA tissue IHC). This supports pattern comparison but does not establish the specificity of an unlisted catalog antibody. |
| Protein features | UniProt reports one chain spanning residues 1–398, no signal peptide or propeptide, no transmembrane segment, and several modified residues (UniProt O95067). The supplied record gives no antibody epitope, so these features do not justify a specific retrieval method or predict phosphosite dependent staining. |
| IF/ICC Q: Where should fluorescence appear? | A: Mainly in the cytosol, with an additional Golgi signal (HPA ICC-IF: cytosol enhanced; Golgi supported). This is an ICC-IF observation; use the cytoplasmic tissue profile to interpret paraffin IHC (HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive cells are blank. | The assay may have failed, or the intended cells may be absent from the section; a blank field alone cannot decide which (general IHC practice). | Confirm the cell type on the counterstained section, then check the catalog antibody's IHC-P instructions, detection reagents, and a positive control (general IHC practice; HPA tissue IHC: High in bone marrow hematopoietic and testis Leydig cells). |
| Most cells stain equally, with little subset contrast. | This differs from HPA's cytoplasmic subset profile; background or nonspecific staining is possible (HPA tissue IHC; general IHC practice). | Compare with a no primary control and review blocking, antibody dilution, washes, and chromogen development under the chosen IHC workflow (general IHC practice). |
| A Not detected cell type stains strongly. | Cross reactivity or endogenous detection activity is possible; HPA's negative observations are reference patterns, not proof of absolute absence (HPA tissue IHC; general IHC practice). | Verify cell identity and inspect a no primary control; if staining remains primary dependent, assess antibody specificity with an independent validation strategy (general IHC practice). |
| Signal appears mainly nuclear or membrane outlined. | That distribution conflicts with the reported cytoplasmic tissue pattern, but localisation alone does not diagnose the artefact (HPA tissue IHC; general IHC practice). | Compare positive tissue and no primary controls, then review counterstain, focus, and detection background before scoring the compartment (general IHC practice). |
| Signal is weak in an expected positive population. | HPA's High category does not prescribe intensity for this antibody, section, or staining run (HPA tissue IHC; general IHC practice). | Check the catalog antibody's IHC-P dilution and retrieval directions, detection performance, and a concurrent positive control; do not infer a CCNB2 specific fixation effect (general IHC practice). |
Experiment evidence
IHC & IF images showing CCNB2 staining
M02040 · IHCIHC analysis of CCNB2 using anti-CCNB2 antibody (M02040). CCNB2 was detected in a paraffin-embedded section of human colon adenocarcinoma ti
M02040 · IHCIHC analysis of CCNB2 using anti-CCNB2 antibody (M02040). CCNB2 was detected in a paraffin-embedded section of human colon adenocarcinoma ti
M02040 · IFImmunofluorescent analysis using the Antibody at 1:50 dilution.
A02040-1 · IHCIHC analysis of CCNB2 using anti-CCNB2 antibody (A02040-1). CCNB2 was detected in paraffin-embedded section of human Ovarian cancer tissue.
A02040-1 · IHCIHC analysis of CCNB2 using anti-CCNB2 antibody (A02040-1). CCNB2 was detected in paraffin-embedded section of human mammary cancer tissue.
A02040-1 · IFIF analysis of CCNB2 using anti-CCNB2 antibody (A02040-1). CCNB2 was detected in immunocytochemical section of U20S cells. Enzyme antigen re
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000157456-CCNB2/tissue/cerebral+cortex#imid_2871356
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000157456-CCNB2/tissue/colon#imid_2871290
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000157456-CCNB2/tissue/liver#imid_2871283
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000157456-CCNB2/tissue/kidney#imid_2871284
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA008873; green = CCNB2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA008873; green = CCNB2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CCNB2 IHC & IF
🧪Run colon first and score its endocrine cells for CCNB2 staining (HPA: High in colon endocrine cells). Run adipose tissue as the negative tissue and score adipocytes for absent staining (HPA: Not detected in adipocytes); on the colon slide, use cells without specific signal as background comparators, but do not assign a particular non-endocrine cell type as a confirmed negative because the supplied HPA row does not identify one.
Positive control tissue: Appendix (Endocrine cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CCNB2 in A-431, U-251MG, U2OS, with annotated localisation: Cytosol (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a concentration-matched rabbit IgG isotype control, and a CCNB2-knockout specimen as a biological negative (M02040 caption: rabbit primary antibody). For colon HRP/DAB staining, quench endogenous peroxidase and check the no-primary slide for residual background (M02040 caption: HRP/DAB detection).
⚠️Feasibility: The selected-SKU paraffin-section caption reports no fixative or target-specific fixation window or effect (M02040 caption: fixative not stated). It uses heat retrieval in EDTA at pH 8.0, but does not establish whether retrieval is required (M02040 caption: heat-mediated EDTA retrieval). Frozen sections are not evaluated in the supplied evidence; IF/ICC images exist for A-431, U-251MG and U2OS, but do not establish that IF is easier for colon tissue (HPA: ICC-IF image cell lines).
HPA tissue IHC evidence for CCNB2
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 |
|---|---|---|---|---|
| Appendix | Endocrine cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Colon | Endocrine cells | High | Protein (IHC) | HPA → |
| Duodenum | Endocrine cells | High | Protein (IHC) | HPA → |
| Rectum | Endocrine cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Lung | Alveolar cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CCNB2 IHC Tips
Use the catalog antibody’s paraffin section result as a starting point, then assess staining against CCNB2’s cell cycle role and reported localisation.
What retrieval should I try first when CCNB2 staining is weak?
Start with heat-mediated retrieval in EDTA at pH 8.0 (datasheet M02040). This is the retrieval used for the catalog antibody’s paraffin section image, followed by a 1:50 primary incubation overnight at 4°C (datasheet M02040). If staining remains weak, check that sections were fully deparaffinised and compare retrieval heating and cooling conditions across matched sections (standard IHC practice). Keep the antibody dilution and detection steps constant while testing retrieval, so changes in signal remain interpretable (standard IHC practice). Confirm any apparent improvement against cellular morphology and a no-primary control before adopting the condition (standard IHC practice).
Could fixation explain weak CCNB2 staining in my paraffin sections?
The selected paraffin section caption does not state its fixative, so target-specific fixation sensitivity is unknown (datasheet M02040). Record the fixative, time in fixative, and tissue processing conditions for each specimen before comparing staining across cases (standard IHC practice). If archival blocks vary, compare sections with similar processing and run the same EDTA pH 8.0 retrieval and 1:50 primary dilution first (datasheet M02040; standard IHC practice). Assess preservation of morphology alongside signal, because damaged or unevenly processed tissue can make staining difficult to interpret (standard IHC practice). Do not attribute a fixation effect to CCNB2 without a controlled comparison (standard IHC practice).
Where should I expect CCNB2 staining in a positive section?
Expect staining in a subset of cells, predominantly in the cytoplasm, when evaluating chromogenic tissue sections (HPA tissue IHC: cytoplasmic expression in a subset of cells). Cytosolic localisation is independently supported by cell imaging, with additional Golgi localisation reported (HPA subcellular: cytosol enhanced; Golgi supported). Because CCNB2 controls the G2/M transition, the fraction of stained cells may vary with the cell cycle composition of the sampled area (UniProt O95067 function; interpretation). Score the compartment and cell population separately rather than treating any brown deposit as equivalent signal (standard IHC practice). Review unexpected exclusively nuclear or diffuse extracellular staining against controls and morphology (HPA localisation; standard IHC practice).
How do isoforms and epitope accessibility affect CCNB2 IHC interpretation?
The supplied record lists 0 isoforms and one 1–398 chain, so it provides no annotated isoform-specific staining pattern to resolve (UniProt O95067 processing and isoforms). It lists no transmembrane segment or signal peptide, which does not establish where this antibody’s epitope lies (UniProt O95067 topology and processing). Several modified residues are recorded, including phosphorylation at positions 8, 11, 77, 92, and 94, but the antibody’s epitope and modification sensitivity are unspecified (UniProt O95067 modified residues; datasheet M02040). If staining changes with retrieval, test matched sections before assigning that change to an isoform or phosphorylation state (standard IHC practice). Interpret the observed compartment using staining controls and the reported cytoplasmic profile (HPA tissue IHC; standard IHC practice).
How should I follow up a CCNB2 IHC pattern with multiplex IF?
Use IF as a separate validation experiment and choose a marker for the cell population you expect to stain; the IHC profile reports high signal in bone marrow hematopoietic cells and in appendix endocrine cells (HPA tissue IHC). Select fluorophores after inspecting tissue autofluorescence, and place the weaker channel away from the strongest background where the available filters permit (standard IF practice). For an intracellular CCNB2 epitope, include a permeabilisation step and optimise it against preserved morphology; the record lists no transmembrane segment, while the antibody’s precise epitope is unspecified (UniProt O95067 topology; standard IF practice). Include single-channel and no-primary controls when evaluating overlap (standard IF practice). Do not transfer the paraffin section’s 1:50 dilution directly to IF without titration (datasheet M02040; standard IF practice).
How can I reduce widespread brown background without losing CCNB2 signal?
First inspect a no-primary section to separate detection background from primary-antibody-associated staining (standard IHC practice). The catalog image used 10% goat serum blocking, a 1:50 rabbit primary overnight at 4°C, a peroxidase-conjugated secondary for 30 minutes at 37°C, and DAB detection (datasheet M02040). Check endogenous peroxidase blocking, washing, and DAB development on matched sections before changing the primary dilution (standard chromogenic IHC practice). If background remains diffuse, titrate primary antibody and detection exposure while retaining a known positive control (standard IHC practice). Distinguish granular cellular signal from pigment, edge deposits, and precipitate under the counterstain (standard IHC practice).
What is a defensible way to quantify heterogeneous CCNB2 staining? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, because tissue IHC reports cytoplasmic staining in a subset of cells (HPA tissue IHC). Record the percentage of positive cells and staining intensity; an H-score can summarise both using 0–3 intensity categories (standard IHC scoring practice). For spatial comparisons, report positive cells per mm² of viable tissue and state the counting method (standard IHC quantification practice). Normalise cell counts to the eligible cell population or viable sampled area, and apply the same positivity threshold across specimens (standard IHC quantification practice). Keep compartment scores separate and review discordant fields for processing artefacts before pooling results (HPA localisation; standard IHC practice).
How do I distinguish genuine CCNB2 positivity from staining artefact?
A credible result should show cellular staining in an appropriate compartment: tissue IHC reports cytoplasmic expression in a subset of cells, and cell imaging supports cytosolic localisation with additional Golgi signal (HPA tissue IHC; HPA subcellular). Compare the stained population with tissue morphology and the expected variation associated with CCNB2’s G2/M role (UniProt O95067 function; standard IHC practice). Treat isolated edge staining, necrotic deposits, and signal present in the no-primary control as possible artefacts (standard IHC practice). Check endogenous peroxidase activity when DAB appears in unexpected structures (standard chromogenic IHC practice). A single negative region does not establish assay failure when the reported tissue pattern is heterogeneous (HPA tissue IHC).
Boster reagents
Best CCNB2 / G2/mitotic-specific cyclin-B2 IHC Antibodies
Both anti-CCNB2 antibodies have human paraffin-section IHC images; A02040-1 also has an IF image of U20S cells (catalog image captions). Both list human, mouse and rat reactivity (catalog).
Real IHC data
Anti-Cyclin B2 CCNB2 Rabbit Monoclonal Antibody
Cat # M02040
Real IHC data
Anti-Cyclin B2/CCNB2 Antibody ®
Cat # A02040-1
M02040 has IHC images of human colon adenocarcinoma and liver cancer paraffin sections, plus IF images with dilution-only captions (catalog image captions). A02040-1 has IHC images of human ovarian and mammary cancer paraffin sections and an IF image of U20S cells (catalog image captions).
Which to pick: For tissue IHC, choose M02040 if a monoclonal antibody is preferred (catalog: clone GIG-3); its own paraffin-section IHC caption uses EDTA retrieval at pH 8.0 and 1:50 primary dilution, with fixative unreported (M02040 IHC caption). For IF/ICC, A02040-1 offers a documented cell-image workflow at 2 μg/mL (A02040-1 IF caption). For cross-species planning, both list human, mouse and rat reactivity, while their IHC images document human tissue only (catalog reactivity; respective IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O95067 (CCNB2_HUMAN, G2/mitotic-specific cyclin-B2).
- Human Protein Atlas. CCNB2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CCNB2 subcellular location (ICC-IF): Mainly localized to the cytosol. In addition localized to the Golgi apparatus..
- Human Protein Atlas. CCNB2 antibody validation summary (2 antibodies).
- Prognostic Significance of CCNB2 Expression in Triple-Negative Breast Cancer. Cancer management and research 2021 — PMC8728388.
- Knockdown of CCNB2 inhibits the tumorigenesis of gastric cancer by regulation of the PI3K/Akt pathway. Scientific reports 2025 — PMC11832750.
- Elevated cyclin B2 expression in invasive breast carcinoma is associated with unfavorable clinical outcome. BMC cancer 2013 — PMC3545739.
- Cyclin B2 (CCNB2) Stimulates the Proliferation of Triple-Negative Breast Cancer (TNBC) Cells In Vitro and In Vivo. Disease markers 2021 — PMC8331305.
- PubMed PMID:11230166 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CCNB2 (G2/mitotic-specific cyclin-B2). Expected localisation Cytoplasmic staining in a subset of cells (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M02040). Validated IHC antibody: M02040 at 1:50 (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Endocrine cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon adenocarcinoma tissue; fixative not specified (datasheet M02040); 2. Fixation: Image fixative and duration unreported (datasheet M02040); 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 retrieval: EDTA pH 8.0 (datasheet M02040); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M02040); 8. Primary antibody: Rabbit monoclonal (clone GIG-3) anti-CCNB2, 1:50 (datasheet M02040); 9. Primary incubation: Overnight at 4 °C (datasheet M02040). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific). Selected-image fixative and duration unreported (datasheet M02040); verify before use.; Caveat: The G2/M cell-cycle role may limit the positive-cell fraction (UniProt); Isoform / epitope: 0 annotated isoforms; one unprocessed chain (UniProt); Regulation: G2/mitotic-specific expression (UniProt). Evidence: UniProt O95067, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the cytosol. In addition localized to the Golgi…, Datasheet M02040, PMC8728388, PMC11832750. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CCNB2/immunohistochemistry-ihc-guide.