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CCNA2 / Cyclin-A2 · IHC design guide
Design Immunohistochemistry for CCNA2
CCNA2 staining is nuclear in a varying fraction of cells across most tissues (HPA tissue IHC). This paraffin IHC guide uses high-staining colon endocrine cells and adipocytes with no detected staining to help plan controls and score positive nuclei (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CCNA2 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 in a varying fraction of tissue cells (HPA tissue IHC) | |
| Staining pattern | Variable fractions of cells show nuclear staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M00700-1) | |
| 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 tissue fixation consistent across samples (standard IHC practice; not target-specific) | |
| Caveat | Cell-cycle dependence may change the positive-cell fraction (UniProt) | |
| Regulation | Linked to G1/S and G2/M transitions (UniProt) | |
| Isoform / epitope | No annotated isoforms; one 1–432 chain (UniProt) |
Section 1
Recommended CCNA2 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet: M00700-1) with one published CCNA2 tissue-chip protocol (PMC11211272: methods).
| Sample | Paraffin-embedded human colorectal adenocarcinoma tissue; fixative not specified (datasheet M00700-1) |
| Fixation | Image fixative and duration unreported (datasheet M00700-1); 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 M00700-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M00700-1) |
| Primary antibody | Rabbit monoclonal (clone HOC-3) anti-CCNA2, 1:50 (datasheet M00700-1) |
| Primary incubation | Overnight at 4 °C (datasheet M00700-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet M00700-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CCNA2-positive staining in endocrine cells of appendix (HPA tissue IHC: High). HPA tissue profile: Nuclear expression in a varying fraction of cells in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet: M00700-1); the published tissue-chip protocol used boiling citric acid in a pressure cooker (PMC11211272: methods).
Section 2
What Is the Expected CCNA2 Staining Pattern?
CCNA2 is predominantly nuclear in tissue IHC, with staining in a varying fraction of cells rather than every cell (HPA tissue IHC). It is exclusively nuclear during interphase, while cytoplasmic localisation can occur at prophase or with SCAPER (UniProt P20248). High staining is reported in gastrointestinal endocrine cells; the HPA tissue profile has Enhanced reliability with medium consistency between antibody staining and RNA expression (HPA tissue IHC). CCNA2 has no transmembrane segment (UniProt P20248 topology).
| Discrete nuclear staining in some cells, with unstained neighbours in the same section (HPA tissue IHC). | This fits the reported nuclear expression in a varying fraction of cells (HPA tissue IHC) and CCNA2's cell cycle role (UniProt P20248). Score the fraction of positive nuclei alongside intensity; do not require uniform staining across the tissue. |
| Predominantly cytoplasmic staining with little or no nuclear signal in an IHC section. | Treat a widespread cytoplasmic-only pattern as a localisation discrepancy against the tissue IHC profile (HPA tissue IHC). Cytoplasmic CCNA2 is possible at prophase or when associated with SCAPER (UniProt P20248); compare morphology and controls before calling the pattern artefactual. |
| Strong staining in a cell population reported as undetected, such as adipocytes (HPA: adipocytes not detected). | Check cell identification and controls. The result differs from the supplied HPA observation; cross-reactivity or endogenous chromogenic activity are possibilities, not diagnoses (general IHC practice). An HPA staining level describes the sampled cells and does not prove that every specimen must match. |
| Diffuse colour across nuclei, cytoplasm, and surrounding tissue, with little cell-to-cell distinction. | This is harder to interpret than discrete nuclear staining (HPA tissue IHC). Examine a no-primary control and review blocking, washes, antibody concentration, and detection chemistry for nonspecific background (general IHC practice). Diffuse colour alone does not establish CCNA2 localisation. |
| No nuclear signal in sampled gastrointestinal endocrine cells reported as high by HPA (HPA tissue IHC). | First confirm that the expected cells are present and assess an appropriate positive control. A negative section could reflect sampling or assay performance (general IHC practice); the HPA observation does not establish a CCNA2-specific fixation effect or guarantee positivity in every specimen. |
💡Expected CCNA2 appearanceCall an IHC result positive when identifiable cells show discrete nuclear staining in a varying fraction of cells (HPA tissue IHC), with high staining reported in gastrointestinal endocrine cells (HPA tissue IHC); widespread cytoplasmic-only colour or uniform diffuse background is suspect and needs controls before interpretation (UniProt P20248; general IHC practice).
| Cell cycle and localisation | CCNA2 controls G1/S and G2/M transitions (UniProt P20248). Interphase localisation is exclusively nuclear, while prophase or SCAPER association can include cytoplasm (UniProt P20248). Interpret an occasional cytoplasmic cell in that context; the tissue IHC profile remains predominantly nuclear (HPA tissue IHC). |
| Tissue and cell selection | HPA reports high staining in endocrine cells of the appendix, colon, duodenum, rectum, and small intestine; breast glandular cells and cervical squamous cells are medium (HPA tissue IHC). Name the cell population being scored, since a tissue-wide average can obscure a varying positive fraction (HPA tissue IHC). |
| Antibody evidence | CAB000114 has IHC Enhanced validation, while HPA020626 has no supplied IHC validation status (HPA antibodies). HPA's tissue profile is Enhanced but has medium staining-to-RNA consistency (HPA tissue IHC). Use the validated IHC pattern as a reference without treating every discrepant cell as definitively negative. |
| Protein architecture and retrieval limits | CCNA2 is a 1–432 chain with no annotated signal peptide, propeptide, glycosylation site, or transmembrane segment (UniProt P20248). These annotations give no CCNA2-specific antigen retrieval setting or fixation sensitivity. Choose and document retrieval conditions through assay optimisation (general IHC practice). |
| IF/ICC Q: Where should fluorescence appear? | A: HPA reports enhanced nucleoplasm and cytosol localisation in ICC-IF (HPA subcellular). UniProt reports nuclear interphase localisation and cytoplasm at prophase or with SCAPER (UniProt P20248). Use the separate IF/ICC guide for that application; this tissue IHC section provides no IF/ICC protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| The expected nuclear signal is absent from a proposed positive section. | The sampled field may lack the relevant cells or the chromogenic assay may have failed (general IHC practice); HPA reports a varying positive fraction (HPA tissue IHC). | Confirm the cell population on the counterstain, inspect a positive control, and review retrieval, antibody incubation, and detection records (general IHC practice). Do not assign a CCNA2-specific fixation cause from these sources. |
| Most colour is cytoplasmic and nuclei remain pale. | The pattern conflicts with predominant tissue nuclear staining (HPA tissue IHC), although cytoplasmic localisation is possible in defined contexts (UniProt P20248). | Compare positive and no-primary controls, inspect cell morphology, and record nuclear and cytoplasmic signal separately (general IHC practice). Avoid calling all cytoplasmic signal specific or all of it artefactual. |
| A reported undetected cell population stains strongly. | Cell misidentification, cross-reactivity, or endogenous detection activity may explain an unexpected result (general IHC practice); HPA lists adipocytes as not detected (HPA tissue IHC). | Verify the cell type, run a no-primary control, and review endogenous peroxidase blocking where relevant to chromogenic detection (general IHC practice). Report the discrepancy against the specified HPA cell population. |
| Diffuse chromogen obscures the boundary of positive nuclei. | Nonspecific reagent binding, inadequate washing, or detection background can reduce contrast (general IHC practice). | Review the no-primary control, blocking and wash steps, antibody concentration, and development time (general IHC practice). Reassess whether distinct nuclear staining remains after background is controlled (HPA tissue IHC). |
| Only a small fraction of otherwise suitable cells stains. | A varying fraction of nuclear-positive cells is expected in most tissues (HPA tissue IHC), consistent with CCNA2's cell cycle role (UniProt P20248). | Record the fraction and intensity in the named cell type, then compare with a positive control and the relevant HPA cell-level observation (HPA tissue IHC; general IHC practice). A low fraction alone does not show assay failure. |
| Signal differs between sections processed in separate IHC runs. | Differences in retrieval, antibody incubation, or chromogen development can alter assay readout (general IHC practice). No target-specific fixation sensitivity is supplied for CCNA2. | Compare run records and controls before interpreting a biological change; standardise the differing workflow step and repeat the comparison if needed (general IHC practice). Keep the expected cell and nuclear pattern anchored to HPA tissue IHC. |
Experiment evidence
IHC & IF images showing CCNA2 staining
M00700-1 · IHCIHC analysis of Cyclin A2 using anti-Cyclin A2 antibody (M00700-1). Cyclin A2 was detected in a paraffin-embedded section of human colorecta
M00700-1 · IHCIHC analysis of Cyclin A2 using anti-Cyclin A2 antibody (M00700-1). Cyclin A2 was detected in a paraffin-embedded section of human liver can
M00700-1 · IFImmunofluorescent analysis using the Antibody at 1:50 dilution.
PB9424 · IHCIHC analysis of Cyclin A2 using anti-Cyclin A2 antibody (PB9424). Cyclin A2 was detected in a paraffin-embedded section of human colorectal
PB9424 · IFIF analysis of Cyclin A2 using anti-Cyclin A2 antibody (PB9424). Cyclin A2 was detected in an immunocytochemical section of A431 cells. Enzy
M00700-3 · IHCHuman colon carcinoma was stained with anti-Cyclin A2 rabbit antibody
M00700-3 · IHCHuman tonsil was stained with anti-Cyclin A2 rabbit antibody
M00700 · IHCImmunohistochemical analysis of paraffin-embedded human colon cancer, using Cyclin A1/A2 Antibody.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000145386-CCNA2/tissue/cerebral+cortex#imid_20043577
HPA · EsophagusHuman Protein Atlas IHC, Esophagus. Source: https://www.proteinatlas.org/ENSG00000145386-CCNA2/tissue/esophagus#imid_20043549
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000145386-CCNA2/tissue/colon#imid_20043503
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000145386-CCNA2/tissue/liver#imid_20043520
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB000114; green = CCNA2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB000114; green = CCNA2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CCNA2 IHC & IF
🧪Run colon first and look for CCNA2 staining in endocrine cells (HPA: High in colon endocrine cells). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes); on the colon slide, cells without convincing nuclear staining serve as internal negative comparators, although the HPA row does not establish that all other cell types are negative.
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 CCNA2 in A-431, U-251MG, U2OS, Rh30, HeLa BAC 5335, HeLa , with annotated localisation: Nucleoplasm (enhanced), Cytosol (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, an isotype control matched to the primary antibody’s host and immunoglobulin class, and a CCNA2 knockout biological negative; the selected caption identifies a rabbit primary antibody (caption: rabbit anti-Cyclin A2). Block endogenous peroxidase before chromogenic detection in colon sections (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the exact selected-SKU tissue-IHC caption does not report a fixative (caption: fixative not stated). The caption used heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required (caption: EDTA retrieval); the supplied evidence does not establish whether frozen sections or IF are easier. Colon endocrine cells may be sparse within the section, so confirm the scored cell type by morphology (HPA: High in colon endocrine cells).
HPA tissue IHC evidence for CCNA2
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 → |
| Colon | Endocrine cells | High | Protein (IHC) | HPA → |
| Duodenum | Endocrine cells | High | Protein (IHC) | HPA → |
| Rectum | Endocrine cells | High | Protein (IHC) | HPA → |
| Small intestine | 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 → |
| 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 CCNA2 IHC Tips
Troubleshoot CCNA2 staining in paraffin sections by checking retrieval, compartment, cell distribution and controls before scoring chromogenic signal.
What retrieval should I try first for weak CCNA2 staining in paraffin sections?
Use heat-mediated EDTA retrieval at pH 8.0 for CCNA2 paraffin-section IHC (datasheet M00700-1). The selected tissue image used this retrieval before an overnight primary incubation at 4°C, so it is the documented starting condition for this antibody (datasheet M00700-1). If nuclear staining is weak, first check that sections were fully deparaffinised and that heating and cooling were consistent across slides (standard IHC practice). Adjust retrieval duration in a controlled comparison while keeping the buffer and detection conditions fixed (standard IHC practice). Compare signal and tissue preservation together; stronger DAB alone does not establish specific CCNA2 staining (standard IHC practice).
How should I troubleshoot fixation when CCNA2 nuclear staining is inconsistent?
The selected CCNA2 tissue caption identifies a paraffin section but does not report its fixative or fixation duration (datasheet M00700-1). Target-specific fixation sensitivity is therefore unknown from the supplied IHC evidence (datasheet M00700-1). Record the fixative and duration for each specimen, then compare sections processed and retrieved together before attributing differences to CCNA2 abundance (standard IHC practice). Include a tissue section with established nuclear staining in each run and inspect morphology for processing damage (HPA: nuclear expression in a varying fraction of cells; standard IHC practice). Do not use the reported localisation or modified residues to infer a fixation requirement (UniProt P20248; standard IHC practice).
Should CCNA2 staining be nuclear, cytoplasmic, or both in tissue IHC?
Expect staining in a varying fraction of nuclei across many tissues, rather than uniform staining of every cell (HPA: tissue IHC profile). CCNA2 is exclusively nuclear during interphase and can appear in both nucleus and cytoplasm at prophase (UniProt P20248, PubMed:1312467). Cytoplasmic localisation is also reported when CCNA2 associates with SCAPER, so a cytoplasmic component warrants cellular and morphological review (UniProt P20248, PubMed:17698606). Compare any cytoplasmic DAB with nuclear signal in the same population and with a control section before accepting it as specific (standard IHC practice). Diffuse staining across unrelated compartments, especially without the expected nuclear pattern, calls for background controls (HPA: tissue IHC profile; standard IHC practice).
Could an isoform or modified epitope explain discordant CCNA2 staining?
The supplied CCNA2 record lists a 432-residue chain and no annotated isoforms, so it offers no documented tissue isoform to explain discordant IHC staining (UniProt P20248). It reports modified residues including phosphoserines at positions 5 and 55, but the antibody epitope and its modification sensitivity are not supplied (UniProt P20248; datasheet M00700-1). Do not interpret a staining difference as phosphorylation-dependent without independent epitope evidence (standard IHC practice). CCNA2 has no transmembrane segment, so a membrane-restricted pattern is not supported by its annotated topology (UniProt P20248). Compare localisation, retrieval consistency and an appropriate control section before assigning a biological explanation (standard IHC practice).
How can IF help resolve an ambiguous chromogenic CCNA2 pattern?
Use IF as a separate localisation check alongside the paraffin-section IHC result; the selected antibody caption documents chromogenic IHC conditions, not IF conditions (datasheet M00700-1). Multiplex CCNA2 with a validated marker for the cell population being assessed, such as an endocrine-cell marker when investigating the HPA-reported colon endocrine-cell signal (HPA: colon endocrine cells, High; standard IF practice). Choose fluorophores after inspecting unstained tissue autofluorescence, favouring a channel with clear separation from that background (standard IF practice). Permeabilise sufficiently to reach nuclear and cytosolic CCNA2, which has no transmembrane segment, while preserving cell boundaries (UniProt P20248; standard IF practice). Evaluate nuclear and cytosolic signal separately against single-stain and secondary-only controls (HPA: nucleoplasm and cytosol; standard IF practice).
How do I separate weak CCNA2 staining from DAB background?
The selected IHC image used 10% goat-serum blocking, a 1:50 rabbit primary incubation overnight at 4°C, and peroxidase-based DAB detection (datasheet M00700-1). Check whether diffuse colour persists in a section processed without primary antibody; that result points to detection-system or tissue background (standard IHC practice). Use an endogenous-peroxidase block before HRP detection and compare DAB development across slides under the same conditions (standard IHC practice). Examine tissue edges, damaged regions and pigment before calling faint brown signal cellular CCNA2 staining (standard IHC practice). Specific staining should remain interpretable as a cellular pattern, particularly in nuclei, rather than diffuse colour across the section (HPA: tissue IHC profile; standard IHC practice).
What is a defensible way to score CCNA2 in chromogenic sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and anatomical region before scoring, because HPA reports nuclear CCNA2 in a varying fraction of cells (HPA: tissue IHC profile; standard IHC practice). Report the percentage of evaluable cells with nuclear DAB and, when intensity is reliably separated, an H-score using the same thresholds across slides (standard IHC practice). For spatial questions, report positive nuclei per mm² of evaluable tissue and state the region sampled (standard IHC practice). Normalise each count to evaluable cells or tissue area, excluding folds, necrosis and missing tissue from the denominator (standard IHC practice). Score cytoplasmic signal separately if investigated, given the reported cell-cycle-dependent localisation (UniProt P20248, PubMed:1312467; standard IHC practice).
When is an unusual CCNA2-positive area more likely artefact than biology?
A convincing result shows cellular staining in an interpretable population, commonly a varying fraction of nuclei in tissue IHC (HPA: tissue IHC profile; standard IHC practice). Strong membrane-only colour conflicts with CCNA2's lack of a transmembrane segment, while cytoplasmic staining requires context because it can occur at prophase or with SCAPER association (UniProt P20248, PubMed:1312467 and PubMed:17698606). Question signal confined to section edges, necrotic areas or visibly damaged tissue, and compare it with intact regions on the same slide (standard IHC practice). Check a no-primary control when widespread DAB suggests endogenous enzyme activity or detection background (standard IHC practice). Interpret unexpected cell populations cautiously: HPA reports high staining in colon endocrine cells but medium consistency between staining and RNA data overall (HPA: tissue IHC profile and reliability description).
Boster reagents
Best CCNA2 / Cyclin-A2 IHC Antibodies
The catalog includes anti-CCNA2 antibodies with IHC images of human tissue and IF images of cells; Human, Mouse and Rat reactivity is listed for selected SKUs (catalog applications, reactivity and image captions).
Real IHC data
Anti-Cyclin A2 Rabbit Monoclonal Antibody
Cat # M00700-1
Real IHC data
Anti-Cyclin A2/CCNA2 Antibody ®
Cat # PB9424
Real IHC data
Anti-Cyclin A2 Rabbit Monoclonal Antibody
Cat # M00700-3
Real IHC data
Anti-Cyclin A1/A2 Rabbit Monoclonal Antibody
Cat # M00700
M00700-1 has IHC images of paraffin-embedded human colorectal adenocarcinoma and listed IF/ICC use; PB9424 has IHC imaging of the same tissue type and IF imaging of A431 cells (respective catalog applications and image captions). M00700-3 has IHC images of human colon carcinoma and tonsil, while M00700 has an IHC image of paraffin-embedded human colon cancer and is labeled Cyclin A1/A2 (respective catalog titles and image captions).
Which to pick: For paraffin-section IHC, choose the Cyclin A2-specific monoclonal M00700-1 over the Cyclin A1/A2-labeled M00700; M00700-1’s image documents EDTA retrieval at pH 8.0 and a 1:50 primary dilution, but reports no fixative (catalog titles; M00700-1 and M00700 IHC captions). For IF/ICC, PB9424 has an A431 cell IF image at 5 μg/mL, while M00700-1 also lists IF/ICC and has IF images (PB9424 IF caption; M00700-1 catalog applications and IF captions). For Mouse or Rat samples, M00700-1 and M00700-3 list both species and monoclonal antibodies, although their supplied IHC images show human samples (respective catalog titles, reactivity and IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P20248 (CCNA2_HUMAN, Cyclin-A2).
- Human Protein Atlas. CCNA2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CCNA2 subcellular location (ICC-IF): Localized to the nucleoplasm and cytosol..
- Human Protein Atlas. CCNA2 antibody validation summary (2 antibodies).
- Multi-omics analysis and experimental validation of the value of monocyte-associated features in prostate cancer prognosis and immunotherapy. Frontiers in immunology 2024 — PMC11211272.
- Integrated Profiling Identifies CCNA2 as a Potential Biomarker of Immunotherapy in Breast Cancer. OncoTargets and therapy 2021 — PMC8043851.
- CCNA2 and CCNB3 as Early Potential Molecular Candidates of Oocyte Maturation in Cumulus-Oophorous Complex Cells from Follicular Fluid. Diagnostics (Basel, Switzerland) 2025 — PMC12563169.
- Molecular Characterization and Clinical Relevance of Lysine Acetylation Regulators in Urological Cancers. Frontiers in oncology 2021 — PMC8202406.
- PubMed PMID:1967822 — UniProt-cited evidence.
- PubMed PMID:8202514 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CCNA2 (Cyclin-A2). Expected localisation Nuclear in a varying fraction of tissue cells (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M00700-1). Validated IHC antibody: M00700-1 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 colorectal adenocarcinoma tissue; fixative not specified (datasheet M00700-1); 2. Fixation: Image fixative and duration unreported (datasheet M00700-1); 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 retrieval: EDTA pH 8.0 (datasheet M00700-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M00700-1); 8. Primary antibody: Rabbit monoclonal (clone HOC-3) anti-CCNA2, 1:50 (datasheet M00700-1); 9. Primary incubation: Overnight at 4 °C (datasheet M00700-1). Watch for: Fixation: Keep tissue fixation consistent across samples (standard IHC practice; not target-specific); Caveat: Cell-cycle dependence may change the positive-cell fraction (UniProt); Isoform / epitope: No annotated isoforms; one 1–432 chain (UniProt); Regulation: Linked to G1/S and G2/M transitions (UniProt). Evidence: UniProt P20248, HPA tissue IHC · Enhanced, HPA ICC-IF · nucleoplasm and cytosol., Datasheet M00700-1, PMC11211272. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CCNA2/immunohistochemistry-ihc-guide.