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AURKA / Aurora kinase A · IHC design guide
Design Immunohistochemistry for AURKA
Plan paraffin-section AURKA IHC with catalog antibody A00246-4 at 2–5 μg/ml (datasheet A00246-4). Use testis pachytene spermatocytes and tonsil germinal center cells as positive controls, and score cytoplasmic and nuclear staining by cell type (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
AURKA 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 and nuclear staining in selected cells (HPA tissue IHC) | |
| Staining pattern | Pachytene spermatocytes and immune cells: cytoplasm and nucleus (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A00246-4) | |
| Positive control | Testis+1 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Whole-tissue scores can miss cell-restricted staining (HPA tissue IHC) | |
| Regulation | High in testis; weak in skeletal muscle (UniProt) | |
| Isoform / epitope | No isoforms annotated; one 1–403 chain, no transmembrane segment (UniProt) |
Section 1
Recommended AURKA IHC & IF Protocols
The catalog antibody’s IHC-P protocol uses heat-mediated EDTA pH 8.0 retrieval (datasheet: A00246-4). The published option describes AURKA staining in FFPE colorectal tumors and liver metastases (PMC8688726).
| Sample | Paraffin-embedded human cervical cancer tissue; fixative not specified (datasheet A00246-4) |
| Fixation | Image fixative and duration unreported (datasheet A00246-4); 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 A00246-4); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A00246-4) |
| Primary antibody | Rabbit anti-AURKA, 2-5 μg/ml (datasheet A00246-4) |
| Primary incubation | Overnight at 4 °C (datasheet A00246-4) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A00246-4) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | AURKA-positive staining in pachytene spermatocytes of testis (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic and nuclear expression most abundant in pachytene spermatocytes and immune cells. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 retrieval for the catalog antibody (datasheet: A00246-4); the published protocol specifies a conditioning solution but gives no formulation (PMC8688726).
Section 2
What Is the Expected AURKA Staining Pattern?
AURKA is an intracellular mitotic kinase associated with centrosomes and spindle poles (UniProt O14965 topology and localization). In tissue IHC, expect cytoplasmic and nuclear staining most abundant in pachytene spermatocytes and immune cells (HPA tissue IHC). HPA rates the tissue profile Enhanced, with medium consistency between antibody staining and RNA expression; interpret faint or unexpected staining alongside controls (HPA tissue IHC).
| Medium cytoplasmic and nuclear staining in testis pachytene spermatocytes or tonsil germinal center cells (HPA tissue IHC). | This matches the reported tissue pattern (HPA tissue IHC). A focal centrosomal signal in a mitotic cell is also plausible from AURKA localization, but a paraffin section may not resolve it (UniProt O14965 localization; general IHC practice). |
| Predominantly extracellular or widespread uniform surface staining, with little signal in the expected intracellular compartments. | That distribution does not match the principal HPA tissue profile or UniProt mitotic localization (HPA tissue IHC; UniProt O14965 localization). Review the negative control and detection conditions before assigning it to AURKA (general IHC practice). |
| Strong staining in adipocytes or adrenal glandular cells, especially when the expected positive cells are unstained. | HPA reports AURKA as not detected in those cell populations (HPA tissue IHC). Consider cross-reactivity or endogenous detection activity, then compare with a no-primary control (general IHC practice). |
| Diffuse chromogen across most of the section, including cells expected to have little or no staining. | A uniform haze obscures the cell-specific pattern reported by HPA (HPA tissue IHC). Check blocking, washes, antibody concentration and detection background using matched controls (general IHC practice). |
| No signal in pachytene spermatocytes or tonsil germinal center cells (HPA tissue IHC). | These are reported medium-staining populations, so an absent result cannot establish a negative sample on its own (HPA tissue IHC). Check section integrity, retrieval, antibody and detection performance with a known-positive section (general IHC practice). |
💡Expected AURKA appearanceCall a result positive when cytoplasmic and nuclear staining is evident at roughly medium intensity in pachytene spermatocytes or tonsil germinal center cells (HPA tissue IHC); diffuse extracellular stain or strong signal in HPA not-detected cell populations calls for control review (HPA tissue IHC; general IHC practice).
| Cell population and mitotic state (HPA tissue IHC; UniProt O14965 localization) | HPA reports medium staining in selected testis and tonsil cells; UniProt places AURKA at centrosomes in mitosis and at spindle poles from prophase to anaphase. Score the named cell population and compartment, not section-wide intensity. |
| Antibody validation (HPA antibodies; HPA tissue IHC) | The overall tissue profile is Enhanced, with medium RNA–staining consistency. The supplied antibody records differ: CAB001454 is IHC Enhanced, while HPA002636 is IHC Approved. Keep the chosen antibody's validation status distinct from the overall profile. |
| Topology and processing (UniProt O14965 topology and processing) | UniProt lists no transmembrane segment or signal peptide and one AURKA chain spanning residues 1–403. An intracellular pattern is therefore the main expectation; these annotations do not predict retrieval conditions or fixation sensitivity. |
| Detection background (general IHC practice) | Endogenous enzyme activity can mimic chromogenic staining when the detection system uses that enzyme. Use the appropriate enzyme-blocking and no-primary controls to distinguish detection background from cell-specific signal; HPA tissue staining does not establish its cause. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive testis or tonsil cells show no stain (HPA tissue IHC). | The result may reflect an unsuccessful IHC run rather than absent AURKA in those reported medium-staining cells (HPA tissue IHC; general IHC practice). | Confirm tissue morphology and positive-control performance; review retrieval, primary-antibody application and detection steps using the antibody's validated IHC-P instructions (general IHC practice). |
| Expected cells stain faintly, while controls otherwise perform. | HPA reports medium, not uniformly strong, staining in pachytene spermatocytes and germinal center cells (HPA tissue IHC). | Compare like cell populations at the same scoring threshold; if the positive control is also weak, check antibody dilution and detection conditions against the validated IHC-P instructions (general IHC practice). |
| Adipocytes or adrenal glandular cells stain strongly (HPA tissue IHC). | HPA records these specific cell populations as not detected; nonspecific binding or endogenous detection activity is possible (HPA tissue IHC; general IHC practice). | Review a matched no-primary control and the detection system's blocking steps before interpreting the staining as AURKA (general IHC practice). |
| Broad haze makes nuclei and cytoplasm hard to score. | Excess detection background, incomplete washing or nonspecific primary binding can obscure the reported cell-specific pattern (HPA tissue IHC; general IHC practice). | Check the no-primary control, wash steps and antibody concentration; evaluate whether the expected positive cells remain distinguishable (general IHC practice). |
| Signal is mainly extracellular or appears as a uniform surface outline. | That pattern diverges from HPA's cytoplasmic and nuclear tissue profile and UniProt's principal centrosome and spindle localization (HPA tissue IHC; UniProt O14965 localization). | Inspect morphology and controls, then compare staining with the expected positive cell populations before scoring; UniProt also lists a basolateral membrane location, so do not dismiss a focal membrane signal solely by compartment (UniProt O14965 localization; general IHC practice). |
| IF/ICC: Where should AURKA fluorescence appear? | HPA lists centrosome and basal body as supported locations; mitotic spindle, nucleoplasm and cytosol carry uncertain location labels (HPA subcellular ICC-IF). | Look for centrosomal or basal-body enrichment and interpret other compartments with the stated HPA uncertainty; use the separate IF/ICC guide for method details (HPA subcellular ICC-IF). |
Experiment evidence
IHC & IF images showing AURKA staining
A00246-4 · IHCIHC analysis of Aurora A/AURKA using anti-Aurora A/AURKA antibody (A00246-4). Aurora A/AURKA was detected in a paraffin-embedded section of
A00246-4 · IHCIHC analysis of Aurora A/AURKA using anti-Aurora A/AURKA antibody (A00246-4). Aurora A/AURKA was detected in a paraffin-embedded section of
A00246-3 · IHCIHC analysis of Aurora A/AURKA using anti-Aurora A/AURKA antibody (A00246-3). Aurora A/AURKA was detected in a paraffin-embedded section of
A00246-3 · IHCIHC analysis of Aurora A/AURKA using anti-Aurora A/AURKA antibody (A00246-3). Aurora A/AURKA was detected in a paraffin-embedded section of
A00246-3 · IFIF analysis of Aurora A/AURKA using anti-Aurora A/AURKA antibody (A00246-3). Aurora A/AURKA was detected in an immunocytochemical section of
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000087586-AURKA/tissue/cerebral+cortex#imid_710112
HPA · CaudateHuman Protein Atlas IHC, Caudate. Source: https://www.proteinatlas.org/ENSG00000087586-AURKA/tissue/basal+ganglia#imid_1253038
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000087586-AURKA/tissue/colon#imid_710054
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000087586-AURKA/tissue/liver#imid_710047
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA002636; green = AURKA, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA002636; green = AURKA, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for AURKA IHC & IF
🧪Run testis first and assess staining in pachytene spermatocytes, which have Medium AURKA staining (HPA: testis, pachytene spermatocytes). Use adipose tissue as the negative tissue; adipocytes are listed as Not detected (HPA: adipose tissue, adipocytes), and compare unstained neighboring cells on the testis slide as an internal background reference without assuming that every other cell type is AURKA-negative.
Positive control tissue: Testis (Pachytene spermatocytes, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show AURKA in A-431, CACO-2, U2OS, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), KOLF2.1J, with annotated localisation: Mitotic spindle (uncertain), Centrosome (supported), Basal body (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a matched rabbit IgG isotype control, matching clonality if known (selected-SKU tissue-IHC caption: rabbit primary antibody; standard IHC practice); use AURKA knockout material, if available, as a biological specificity control (standard IHC practice). Quench endogenous peroxidase for HRP/DAB detection and check tissue autofluorescence if adapting the assay to IF (selected-SKU tissue-IHC caption: HRP/DAB; standard IHC/IF practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence, and the selected-SKU paraffin-section caption does not report a fixative (selected-SKU tissue-IHC caption). The caption documents heat retrieval in EDTA at pH 8.0 for paraffin-section IHC, but does not establish that retrieval is required; the supplied evidence does not establish whether frozen sections or IF are easier (selected-SKU tissue-IHC caption). In testis, densely packed spermatogenic cells may make punctate centrosomal staining difficult to assign to individual cells, so assess localization with cell morphology and the controls above (HPA: testis, pachytene spermatocytes; UniProt O14965: centrosomal localization).
HPA tissue IHC evidence for AURKA
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 |
|---|---|---|---|---|
| Testis | Pachytene spermatocytes | Medium | Protein (IHC) | HPA → |
| Tonsil | Germinal center cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced AURKA IHC Tips
Use the catalog antibody’s paraffin section evidence as the starting point, then assess AURKA staining by cell type, mitotic stage and compartment.
What retrieval should I start with if AURKA staining is weak in paraffin sections?
Start with heat mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A00246-4). The selected antibody’s cervical cancer section was processed this way before incubation at 2 μg/ml overnight at 4°C (caption A00246-4). If staining remains weak, vary heating duration on matched sections while holding antibody concentration and detection conditions constant (standard IHC practice). Include a positive control containing pachytene spermatocytes or tonsil germinal center cells to distinguish retrieval failure from a low abundance sample (HPA tissue IHC). Score cellular staining against background rather than treating stronger DAB deposition alone as improved specificity (standard IHC practice).
Could fixation explain weak or uneven AURKA staining?
The selected paraffin section caption does not state a fixative, so AURKA sensitivity to fixation in this IHC workflow is unknown (caption A00246-4). Record fixative, fixation duration and section processing for each sample, and compare matched sections before changing retrieval or antibody concentration (standard IHC practice). Overfixation can reduce antigen accessibility in general, while uneven fixation can produce spatially variable staining; neither effect has been established specifically for this antibody (standard IHC practice; caption A00246-4). Use the documented EDTA pH 8.0 retrieval as the starting condition (datasheet A00246-4). Interpret weak staining alongside a processed positive control and tissue morphology (HPA tissue IHC; standard IHC practice).
Which AURKA staining patterns should I expect across the cell cycle?
Look for centrosomal staining in mitotic cells from early prophase through telophase and spindle pole staining from prophase through anaphase (UniProt O14965 localisation). AURKA can move to the midbody during telophase and cytokinesis, so score those structures separately when morphology permits (UniProt O14965 localisation). In tissue IHC, HPA reports cytoplasmic and nuclear staining most abundant in pachytene spermatocytes and immune cells; those broader compartments have different evidential meaning from a resolved mitotic structure (HPA tissue IHC; HPA subcellular). Check the counterstained nucleus and mitotic morphology before assigning a focal DAB deposit to a centrosome (standard IHC practice). Record the cell type and compartment for each scoring category (standard IHC practice).
Can an isoform or phosphorylation explain a changed IHC pattern?
The supplied record lists 0 isoforms and one AURKA chain spanning residues 1–403, so it provides no annotated isoform explanation for discrepant staining (UniProt O14965 processing and isoforms). AURKA has reported phosphorylation sites, including threonines 287 and 288, but the catalog antibody’s binding epitope and phosphorylation dependence are not supplied (UniProt O14965 modified residues; caption A00246-4). Do not label a staining change as altered phosphorylation without an independently validated site specific assay (standard IHC practice). Compare retrieval and staining conditions on adjacent sections first (standard IHC practice). Document whether the discrepancy concerns cell type, compartment or signal intensity before investigating epitope accessibility (standard IHC practice).
How can I use IF to clarify ambiguous AURKA localisation?
On the separate IF/ICC guide, multiplex AURKA with a marker for the expected cell type, such as a germ cell or immune cell marker selected and validated for the specimen (HPA tissue IHC; standard IF practice). Choose fluorophores and exposure settings after examining unstained tissue autofluorescence, and include single label controls to assess channel bleed through (standard IF practice). AURKA has no transmembrane segment and is reported at intracellular centrosomes, spindle poles and basal bodies, so use permeabilisation compatible with an intracellular epitope (UniProt O14965 topology and localisation; standard IF practice). The antibody epitope is unspecified, so optimise permeabilisation empirically without assuming a mapped epitope location (caption A00246-4; standard IF practice).
How do I separate AURKA signal from diffuse DAB background?
Compare stained sections with a no primary control and inspect whether DAB follows tissue edges, folds or damaged areas (standard IHC practice). The selected paraffin section used 10% goat serum blocking, a peroxidase conjugated anti rabbit secondary and DAB development (caption A00246-4). Apply an endogenous peroxidase block and check detection only controls when diffuse or blood associated deposits persist; these are general chromogenic workflow checks (standard IHC practice). Titrate the primary around the documented 2 μg/ml condition on matched sections while keeping retrieval and development time fixed (caption A00246-4; standard IHC practice). Retain compartment and cell type information when judging specificity (UniProt O14965 localisation; HPA tissue IHC).
How should I quantify AURKA across heterogeneous tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define regions and cell types before scoring, then report the percentage of positive cells and, where intensity is reproducible, an H-score (standard IHC practice). For sparse mitotic structures, count positive cells per mm² of viable tissue and record mitotic cells separately (UniProt O14965 function and localisation; standard IHC practice). Normalise cell counts to the number of eligible cells or sampled viable area, using the same rule across sections (standard IHC practice). Keep centrosomal, spindle associated, cytoplasmic and nuclear scores separate because AURKA localisation changes through mitosis and broader tissue staining is also reported (UniProt O14965 localisation; HPA tissue IHC). Exclude necrosis, folds and section edges using predefined criteria (standard IHC practice).
When is an AURKA positive IHC result convincing?
A convincing result combines the expected cell context with interpretable intracellular staining and low signal in detection controls (HPA tissue IHC; UniProt O14965 localisation; standard IHC practice). Pachytene spermatocytes and tonsil germinal center cells offer reported positive contexts, while adipocytes are reported as undetected in the listed adipose tissue sample (HPA tissue IHC). In mitotic cells, centrosomal or spindle pole staining supports the assignment, although chromogenic resolution may limit identification of these small structures (UniProt O14965 localisation; standard IHC practice). Treat staining restricted to edges, necrotic areas or endogenous enzyme rich deposits as suspect until controls resolve it (standard IHC practice). Report nuclear or diffuse cytoplasmic staining with cell type and control results rather than assigning it to a mitotic structure (HPA tissue IHC; standard IHC practice).
Boster reagents
Best AURKA / Aurora kinase A IHC Antibodies
The catalog includes human paraffin-section IHC images and a Caco-2 cell IF image (catalog image captions: A00246-3, A00246-4); a separate antibody lists human and mouse reactivity (catalog: M00246).
Real IHC data
Anti-Aurora A/AURKA Antibody ®
Cat # A00246-4
Real IHC data
Anti-Aurora A/AURKA Antibody ®
Cat # A00246-3
A00246-4 has IHC images from human cervical, breast, ovarian and squamous cell carcinomas in paraffin sections (catalog IHC captions: A00246-4). A00246-3 has paraffin-section IHC images from human liver cancer and Hashimoto thyroiditis, plus an IF image from Caco-2 cells (catalog IHC/IF captions: A00246-3).
Which to pick: For tissue IHC, choose A00246-4 if its broader set of paraffin-section examples matches your study; its caption reports EDTA pH 8.0 heat retrieval, while the fixative is unreported (catalog IHC captions: A00246-4). For IF/ICC, choose A00246-3, which lists both applications and has a Caco-2 IF image (catalog applications and IF caption: A00246-3). For human and mouse IF/ICC, consider rabbit monoclonal M00246; it lists both species and applications but no IHC validation (catalog: M00246).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O14965 (AURKA_HUMAN, Aurora kinase A).
- Human Protein Atlas. AURKA tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. AURKA subcellular location (ICC-IF): Mainly localized to the centrosome, mitotic spindle and basal body. In addition localized to the nucleoplasm and cytosol..
- Human Protein Atlas. AURKA antibody validation summary (2 antibodies).
- Integrated transcriptomic and immunoinformatics analysis identifies tpx2, bub1b, and ube2c as diagnostic biomarkers and therapeutic targets in endometrial carcinoma. Discover oncology 2025 — PMC12559507.
- Clinical analysis of 10 cases with subcutaneous panniculitis-like T-cell lymphoma and tissue AURKA expression. Skin research and technology : official journal of International Society for Bioengineering and the Skin (ISBS) [and] International Society for Digital Imaging of Skin (ISDIS) [and] International Society for Skin Imaging (ISSI) 2024 — PMC11305869.
- Aurora kinase A (AURKA) expression in colorectal cancer liver metastasis is associated with poor prognosis. British journal of cancer 2013 — PMC3817339.
- Disease Modeling on Tumor Organoids Implicates AURKA as a Therapeutic Target in Liver Metastatic Colorectal Cancer. Cellular and molecular gastroenterology and hepatology 2022 — PMC8688726.
- PubMed PMID:9153231 — UniProt-cited evidence.
- PubMed PMID:9514916 — UniProt-cited evidence.
- PubMed PMID:9771714 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for AURKA (Aurora kinase A). Expected localisation Cytoplasmic and nuclear staining in selected cells (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A00246-4). Validated IHC antibody: A00246-4 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Pachytene spermatocytes); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human cervical cancer tissue; fixative not specified (datasheet A00246-4); 2. Fixation: Image fixative and duration unreported (datasheet A00246-4); 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 A00246-4); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A00246-4); 8. Primary antibody: Rabbit anti-AURKA, 2-5 μg/ml (datasheet A00246-4); 9. Primary incubation: Overnight at 4 °C (datasheet A00246-4). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Whole-tissue scores can miss cell-restricted staining (HPA tissue IHC); Isoform / epitope: No isoforms annotated; one 1–403 chain, no transmembrane segment (UniProt); Regulation: High in testis; weak in skeletal muscle (UniProt). Evidence: UniProt O14965, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the centrosome, mitotic spindle and basal body.…, Datasheet A00246-4, PMC8688726. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/AURKA/immunohistochemistry-ihc-guide.