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CCNA2 / Cyclin-A2 · IHC design guide
Design Immunohistochemistry for CCNA2
Plan CCNA2 chromogenic IHC around nuclear staining in a varying fraction of cells (HPA tissue IHC). This guide covers consistent fixation (standard IHC practice), cell-cycle localisation (UniProt), and the catalog antibody’s 1:50 IHC dilution (datasheet M00700-1).
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 | Nuclear staining in a varying fraction of cells in most tissues (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 | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M00700-1) | |
| Caveat | Prophase can shift signal from nucleus to cytoplasm (UniProt) | |
| Regulation | Staining varies with the cycling-cell fraction (UniProt) | |
| Isoform / epitope | No annotated isoforms; one full-length chain (UniProt) |
Section 1
Recommended CCNA2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: M00700-1) is followed by published CCNA2 protocols for prostate tissue chips (PMC11211272) and follicular-fluid cell smears (PMC12563169).
| 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 pH 8.0 retrieval for paraffin sections (datasheet: M00700-1). A prostate tissue-chip study used pressure-cooker citric acid retrieval (PMC11211272).
Section 2
What Is the Expected CCNA2 Staining Pattern?
CCNA2 staining should be predominantly nuclear in a varying fraction of cells across most tissues (HPA tissue IHC; UniProt P20248: interphase localisation). High staining is reported in gastrointestinal endocrine cells, while several other listed cell populations are not detected (HPA tissue IHC). The tissue IHC profile has Enhanced reliability, with medium consistency between antibody staining and RNA expression (HPA tissue IHC). CCNA2 has no transmembrane segment (UniProt P20248 topology).
| Distinct nuclear staining appears in only some cells; appendix, colon, duodenum, rectum or small-intestine endocrine cells stain strongly. | This fits the variable nuclear fraction reported across tissues and the High level reported in those endocrine populations (HPA tissue IHC). Score the stained cell population and nuclear compartment, rather than treating unstained neighbours as a failed run (HPA tissue IHC; UniProt P20248: interphase localisation). |
| Broad, strong cytoplasmic staining dominates cells whose nuclei show little or no signal. | That pattern warrants an artefact check because CCNA2 is exclusively nuclear during interphase (UniProt P20248). Cytoplasmic signal is possible at prophase or with SCAPER, and ICC-IF also reports cytosol; judge it with cell context and controls (UniProt P20248; HPA subcellular ICC-IF). |
| Adipocytes or liver cholangiocytes stain strongly when the intended positive cells do not. | HPA reports CCNA2 as Not detected in those cell populations (HPA tissue IHC). Consider antibody cross-reactivity or endogenous chromogenic activity, then compare a known-positive section and detection controls (HPA tissue IHC; general IHC practice). |
| Colour spreads across stroma, lumina or many unrelated cells without clear nuclear boundaries. | This is difficult to score as CCNA2 because the tissue profile describes nuclear staining in a varying fraction of cells (HPA tissue IHC). Diffuse deposit can arise from nonspecific binding or detection background; inspect the no-primary control (general IHC practice). |
| No nuclear signal appears in appendix endocrine cells, despite an otherwise readable section. | Those cells are reported High and offer a useful positive comparison, though HPA levels are observations rather than a guarantee for every section (HPA tissue IHC). Check that the population is present, then review assay controls and detection steps (general IHC practice). |
💡Expected CCNA2 appearanceCall a positive result when a subset of appropriate cells shows distinct nuclear chromogen, potentially strong in gastrointestinal endocrine cells; widespread diffuse colour or dominant cytoplasm in interphase cells is suspect (HPA tissue IHC; UniProt P20248: interphase localisation; general IHC practice).
| Cell-cycle localisation | Interphase CCNA2 is exclusively nuclear; nucleus and cytoplasm are reported at prophase, and association with SCAPER can retain CCNA2 in cytoplasm (UniProt P20248). Interpret occasional cytoplasmic cells in that context rather than requiring every stained cell to look identical. |
| Tissue and cell population | HPA reports High staining in endocrine cells of appendix, colon, duodenum, rectum and small intestine; breast glandular cells and endometrial stromal cells are Medium (HPA tissue IHC). The observed fraction and compartment matter more than a whole-section average. |
| Antibody validation | CAB000114 has Enhanced IHC validation; HPA020626 has no IHC status in the supplied record (HPA antibodies). Enhanced reflects reproduction by independent antibodies or orthogonal data, while the tissue profile still reports medium RNA–staining consistency (HPA antibodies; HPA tissue IHC). |
| Processing and topology | UniProt lists one CCNA2 chain spanning residues 1–432, with no signal peptide, propeptide, transmembrane segment or annotated isoforms (UniProt P20248). These annotations give no basis to expect a secreted or membrane staining pattern; epitope-specific retrieval behaviour is unreported. |
| IF/ICC: What pattern should I expect? | HPA reports enhanced nucleoplasm and cytosol localisation in ICC-IF (HPA subcellular ICC-IF). This supports an IF localisation check, while paraffin-section interpretation should follow the tissue IHC pattern and the cell-cycle context (HPA tissue IHC; UniProt P20248). |
| Situation | Likely cause | Next action |
|---|---|---|
| No nuclear staining in a section containing appendix endocrine cells. | A missed assay step or insufficient staining is possible; the cell population is reported High, but its presence alone does not validate a run (HPA tissue IHC; general IHC practice). | Verify the cells are present, compare a concurrent positive section, and check antigen retrieval, primary incubation and detection against the validated IHC workflow (general IHC practice). |
| Strong cytoplasmic colour appears in nearly every cell, with little nuclear definition. | The distribution conflicts with interphase nuclear localisation; prophase or SCAPER-associated cytoplasm may explain individual cells, not an automatic whole-section call (UniProt P20248). | Compare nuclear counterstain and cell context, then inspect no-primary and detection controls for diffuse background (general IHC practice). |
| Adipocytes stain while the intended nuclear positive population does not. | HPA reports adipocytes as Not detected; nonspecific antibody staining or endogenous detection activity is possible (HPA tissue IHC; general IHC practice). | Run a no-primary control and a known-positive tissue, then review blocking and endogenous-peroxidase control for the chromogenic method (general IHC practice). |
| Colour covers tissue broadly and obscures individual nuclei. | Diffuse deposit is inconsistent with a clearly scoreable, variable nuclear pattern (HPA tissue IHC); excess background can result from nonspecific binding or detection conditions (general IHC practice). | Check the no-primary control, review blocking and washes, and adjust staining conditions within the validated assay (general IHC practice). |
| A few cells show both nuclear and cytoplasmic staining. | Nuclear and cytoplasmic CCNA2 is reported at prophase, and cytoplasmic localisation can occur with SCAPER (UniProt P20248). HPA ICC-IF also reports nucleoplasm and cytosol (HPA subcellular ICC-IF). | Record the compartments and frequency, compare adjacent interphase-appearing cells, and avoid calling isolated cytoplasmic signal artefact without controls (UniProt P20248; general IHC practice). |
| Different antibody preparations give different IHC patterns. | The supplied validation is antibody-specific: CAB000114 is IHC Enhanced, while HPA020626 has no supplied IHC status (HPA antibodies). | Anchor interpretation to an IHC-validated antibody, its controls and the reported nuclear tissue pattern; investigate discordant staining before scoring it as CCNA2 (HPA antibodies; HPA tissue IHC; general IHC practice). |
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 appendix first and look for staining in its endocrine cells, which HPA rates High; predominantly nuclear signal is expected in interphase (HPA: High in appendix endocrine cells; UniProt P20248: exclusively nuclear during interphase). Run adipose tissue as the negative tissue, focusing on adipocytes rated Not detected; on the appendix slide, adjacent cells without specific signal can serve as internal background comparators (HPA: Not detected in adipocytes; standard IHC practice).
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 slide; a host- and isotype-matched control IgG; and a CCNA2 knockout specimen or validated peptide-block control where available (standard IHC practice). Quench endogenous peroxidase before HRP/DAB detection, especially where appendix leukocytes could contribute background (standard IHC practice; selected-SKU caption: HRP/DAB detection).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected M00700-1 paraffin-section caption does not state a fixative (selected-SKU caption: fixative not stated). The caption uses heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required (selected-SKU caption: EDTA pH 8.0). The supplied evidence does not establish whether frozen sections or IF/ICC are easier; for IF/ICC, HPA reports nucleoplasmic and cytosolic localisation, and tissue autofluorescence should be checked with an unstained section (HPA: nucleoplasm and cytosol; standard IF practice).
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 by checking retrieval, cellular compartment, cell cycle context, and the source of background before interpreting positive cells.
What retrieval should I try when CCNA2 staining is weak in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for this catalog antibody (datasheet M00700-1). The selected paraffin-section image used that retrieval before overnight incubation with primary antibody at 1:50 and 4°C (caption M00700-1). If staining remains weak, compare retrieval duration or a different buffer on matched sections while holding antibody concentration and detection constant (standard IHC practice). Check section integrity and staining in an expected positive compartment before increasing retrieval intensity, since excessive heating can damage morphology (standard IHC practice; HPA: nuclear expression in a varying fraction of cells). Record each condition alongside its negative control so stronger background is not mistaken for improved CCNA2 detection (standard IHC practice).
How should I troubleshoot inconsistent CCNA2 staining after fixation?
The selected tissue caption identifies a paraffin-embedded section but does not state its fixative, so CCNA2-specific fixation sensitivity is unknown for this antibody (caption M00700-1). Document the fixative and time in fixative for each specimen, then compare similarly processed sections before changing antibody conditions (standard IHC practice). On matched material, repeat the documented EDTA pH 8.0 heat retrieval and 1:50 primary incubation overnight at 4°C (caption M00700-1). Assess whether nuclear signal and morphology vary together across processing batches; fixation and processing differences can change epitope accessibility in paraffin sections (standard IHC practice). Do not infer a CCNA2-specific fixation effect from its phosphorylation sites or tissue distribution (UniProt P20248: modified residues; HPA: tissue IHC profile).
Should cytoplasmic CCNA2 staining count as a positive IHC result?
Score nuclear staining as the primary interphase pattern because CCNA2 is exclusively nuclear during interphase (UniProt P20248; PubMed:1312467). Cytoplasmic staining can be biologically plausible at prophase or when CCNA2 associates with SCAPER, so evaluate those cells separately rather than merging compartments into one score (UniProt P20248; PubMed:1312467, PubMed:17698606). Compare the stained cell's morphology and nuclear counterstain with adjacent cells, and note whether cytoplasmic signal is restricted to a plausible cell population (standard IHC practice). Diffuse cytoplasmic color across many cell types, especially alongside weak nuclear signal, warrants a primary-omission control and review of blocking and detection (standard IHC practice; HPA: nuclear expression in a varying fraction of cells).
Could an isoform or altered epitope explain patchy CCNA2 IHC staining?
The supplied CCNA2 record lists 0 isoforms and a single chain spanning residues 1–432, so an annotated isoform does not explain patchy staining here (UniProt P20248). It lists phosphorylation at serines 5 and 55 and acetylation at methionine 1, but the antibody's epitope is not supplied (UniProt P20248; caption M00700-1). Without epitope mapping, do not attribute a staining difference to any particular modification (standard IHC interpretation). Compare serial sections under the same EDTA pH 8.0 retrieval and primary incubation conditions, and review local cell-cycle context before changing the protocol (caption M00700-1; UniProt P20248: cell-cycle function).
How can IF help resolve ambiguous CCNA2 staining in tissue?
Use IF as a separate validation experiment, pairing CCNA2 with a marker for the expected cell population, such as an endocrine-cell marker in colon, where endocrine cells show high CCNA2 tissue staining (HPA: Colon endocrine cells, High; standard IF practice). Choose fluorophores after inspecting tissue autofluorescence and include single-color controls to identify bleed-through (standard IF practice). CCNA2 has no transmembrane segment and is reported in the nucleus and cytoplasm, so permeabilisation must allow access to intracellular epitopes, including the nucleus when nuclear signal is being assessed (UniProt P20248 topology and subcellular location; standard IF practice). Because the antibody epitope is unspecified, optimize permeabilisation on control material rather than assuming a particular membrane-facing epitope (caption M00700-1; standard IF practice).
What should I check when DAB background obscures CCNA2-positive nuclei?
The selected tissue image used 10% goat serum blocking, a peroxidase-conjugated secondary, and DAB development (caption M00700-1). Check a primary-omission section to distinguish secondary or detection background from primary-dependent staining, and include a peroxidase block before chromogenic development as a general IHC control (standard IHC practice). If background persists, compare blocking, washes, primary concentration, and DAB development on matched sections while preserving the documented EDTA pH 8.0 retrieval (standard IHC practice; caption M00700-1). Judge improvement by discrete nuclear staining in a subset of cells rather than by overall brown intensity, because the tissue profile describes nuclear expression in a varying fraction of cells (HPA: tissue IHC profile).
How should I quantify CCNA2-positive cells across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and scoring area before counting, and use the same thresholds, counterstain, and field-selection rules for every section (standard IHC quantification practice). Report the percentage of nuclei positive for CCNA2 among evaluable nuclei in that population; record cytoplasmic staining separately when present (UniProt P20248: interphase nuclear localisation and prophase cytoplasmic detection; standard IHC quantification practice). If intensity matters, calculate an H-score from the percentages at each intensity level, using a fixed scale and blinded review where feasible (standard IHC quantification practice). For spatial comparisons, report positive nuclei per mm² of viable, analyzable tissue and state that area denominator alongside the cell-based denominator (standard IHC quantification practice).
How can I distinguish genuine CCNA2 positivity from staining artefact?
Look first for discrete nuclear staining in a subset of intact cells, consistent with the reported tissue pattern and CCNA2's interphase localisation (HPA: nuclear expression in a varying fraction of cells; UniProt P20248; PubMed:1312467). Check cell identity against the tissue context: endocrine cells in colon are reported as high, whereas liver cholangiocytes are reported as not detected (HPA: Colon endocrine cells, High; HPA: Liver cholangiocytes, Not detected). Treat staining confined to tissue edges or necrotic areas as suspect and inspect a primary-omission section for endogenous enzyme or detection signal (standard IHC practice). Assess cytoplasmic staining separately because it is reported at prophase and with SCAPER association, while widespread cytoplasmic color alone needs further validation (UniProt P20248; PubMed:1312467, PubMed:17698606; standard IHC practice).
Boster reagents
Best CCNA2 / Cyclin-A2 IHC Antibodies
Catalog antibodies cover human paraffin-section IHC, including colorectal adenocarcinoma, and IF/ICC in A431 cells (catalog image captions); several list human, mouse and rat reactivity (catalog reactivity).
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 and PB9424 each show IHC in human paraffin-embedded colorectal adenocarcinoma; PB9424 also shows IF/ICC in A431 cells (respective image captions). M00700-3 shows IHC in human colon carcinoma and tonsil, while M00700 shows IHC in paraffin-embedded human colon cancer and recognizes Cyclin A1/A2 (respective image captions; M00700 catalog title).
Which to pick: For CCNA2 tissue IHC, start with M00700-1: it is a rabbit monoclonal with human, mouse and rat reactivity, and its own human paraffin-section image specifies EDTA retrieval at pH 8.0 and 1:50 primary dilution (M00700-1 catalog; M00700-1 IHC caption). For IF/ICC, PB9424 has an A431 cell image at 5 μg/mL; M00700-1 also lists IF/ICC, and M00700-3 lists IF with human, mouse and rat reactivity (PB9424 IF caption; M00700-1 catalog; M00700-3 catalog). For cross-species IHC, M00700-1 and M00700-3 list human, mouse and rat reactivity, but their supplied IHC images show human samples; fixation is unreported in those captions, so establish retrieval and dilution for each specimen (respective catalog 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: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M00700-1); Caveat: Prophase can shift signal from nucleus to cytoplasm (UniProt); Isoform / epitope: No annotated isoforms; one full-length chain (UniProt); Regulation: Staining varies with the cycling-cell fraction (UniProt). Evidence: UniProt P20248, HPA tissue IHC · Enhanced, HPA ICC-IF · nucleoplasm and cytosol., Datasheet M00700-1, PMC11211272, PMC12563169. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CCNA2/immunohistochemistry-ihc-guide.