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SKA3 / Spindle and kinetochore-associated protein 3 · IHC design guide
Design Immunohistochemistry for SKA3
Plan chromogenic IHC for SKA3 in paraffin sections using the catalog antibody's 2–5 μg/ml range (datasheet A07561-1). Assess cytoplasmic staining in testis and epididymis, interpreting intensity cautiously because staining and RNA show low concordance (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
SKA3 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 tissue (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic in testis and epididymal glandular cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A07561-1) | |
| Positive control | Epididymis+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 | Staining and RNA show low concordance (HPA tissue IHC) | |
| Regulation | Expression regulation unreported (UniProt) | |
| Isoform / epitope | 3 isoforms; epitope coverage unknown (UniProt) |
Section 1
Recommended SKA3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: A07561-1) is accompanied by published SKA3 protocols for pancreatic adenocarcinoma (PMC11120893) and hepatocellular carcinoma (PMC6895034).
| Sample | Paraffin-embedded human testis tissue; fixative not specified (datasheet A07561-1) |
| Fixation | Image fixative and duration unreported (datasheet A07561-1); 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 A07561-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A07561-1) |
| Primary antibody | Rabbit anti-SKA3, 2-5 μg/ml (datasheet A07561-1) |
| Primary incubation | Overnight at 4 °C (datasheet A07561-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A07561-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | SKA3-positive staining in glandular cells of epididymis (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in testis and epididymis. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet: A07561-1); the included articles do not specify retrieval conditions (PMC11120893; PMC6895034).
Section 2
What Is the Expected SKA3 Staining Pattern?
SKA3 is a non-membrane protein associated with the outer kinetochore and spindle microtubules during mitosis (UniProt Q8IX90: topology and subcellular location). In tissue IHC, expect cytoplasmic staining in testicular pachytene spermatocytes and epididymal glandular cells (HPA: High in both cell types; cytoplasmic profile). Treat this as a provisional reference pattern: HPA rates the IHC staining Approved but reports low consistency with RNA expression and pending external verification (HPA: reliability description).
| Strong cytoplasmic chromogen in pachytene spermatocytes or epididymal glandular cells, with recognizable tissue structure. | This matches the reported tissue pattern (HPA: High in both cell types; cytoplasmic profile). Score the named cells and their compartment, rather than calling an entire section positive; HPA's Approved rating carries a low RNA-consistency caveat and pending external verification (HPA: reliability description). |
| Signal concentrated at a cell-surface rim, in nuclei throughout the section, or in another compartment without the expected cytoplasmic pattern. | Reassess specificity and slide artefact before scoring it as SKA3 (HPA: cytoplasmic tissue profile; UniProt Q8IX90: no transmembrane segment). Mitotic kinetochore or spindle association is biologically plausible (UniProt Q8IX90: subcellular location), so assess compartment together with cell morphology and mitotic state. |
| Strong staining in a cell population reported as undetected, such as adipocytes in adipose tissue. | Consider cross-reactivity or endogenous detection activity and check controls before interpreting it as SKA3 (HPA: adipose adipocytes Not detected; standard IHC practice). An HPA negative is an observed staining result for its tested material, not proof that every specimen must remain negative (HPA: tissue IHC profile). |
| Diffuse color covers stroma, empty spaces, or many unrelated cell types and obscures boundaries. | Treat this as background until controls support a cell-specific pattern (standard IHC practice). Review blocking, washes, chromogen development, and detection controls (standard IHC practice); the reported SKA3 tissue pattern identifies particular cells, not uniform section-wide staining (HPA: tissue IHC profile). |
| No signal in testicular pachytene spermatocytes or epididymal glandular cells. | First verify the named cells are present and the assay's positive control develops (HPA: High in both cell types; standard IHC practice). Then review the catalog antibody's IHC-P instructions and reagent performance (standard IHC practice). HPA supplies no target-specific fixation sensitivity for this result (HPA: tissue IHC profile). |
💡Expected SKA3 appearanceA credible positive shows High cytoplasmic staining in pachytene spermatocytes or epididymal glandular cells (HPA: tissue IHC profile); a uniform membrane rim or widespread staining of HPA-undetected cells is suspect (UniProt Q8IX90: no transmembrane segment; HPA: negative tissue entries).
| Cell cycle and compartment | SKA3 reaches the outer kinetochore and spindle microtubules during mitosis (UniProt Q8IX90: subcellular location). Interpret a mitotic focus in its cellular context; the reported paraffin-tissue pattern is cytoplasmic in testis and epididymis (HPA: tissue IHC profile). |
| Tissue selection and confidence | Use testis or epididymis as pattern references (HPA: High in specified cells), while recording HPA's low staining–RNA consistency and pending external verification (HPA: reliability description). Bone-marrow hematopoietic cells were Not detected by IHC despite tissue-enhanced bone-marrow RNA (HPA: tissue IHC and RNA specificity). |
| Antibody validation | HPA039272 is Approved for IHC; that rating does not remove the tissue-profile caveat (HPA: antibody validation; reliability description). Match the interpretation to the antibody and application used, and use appropriate assay controls (standard IHC practice). |
| IF/ICC Q&A: What pattern should be checked? | Check centrosome and mitotic-spindle signal, with possible cytosolic signal (HPA: subcellular summary). HPA marks those locations uncertain; HPA061534 has Supported ICC validation, whereas HPA039356 is Uncertain (HPA: subcellular locations; antibody validation). This IF/ICC observation does not establish a paraffin-IHC protocol (HPA: application-specific validation). |
| Isoforms and antibody epitope | Three isoforms are listed (UniProt Q8IX90: isoforms 1–3). Without an epitope map in the supplied evidence, do not assume that a given antibody recognizes all three or that isoform choice explains an absent stain (UniProt Q8IX90: isoform record). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive cells are blank. | The named cells may be absent from the section, or the IHC run may have failed (HPA: High in specified cells; standard IHC practice). | Confirm cell identity and inspect the run's positive control; then check the catalog antibody's IHC-P instructions, retrieval conditions, detection reagents, and counterstain (standard IHC practice). |
| All tissue compartments show diffuse color. | Nonspecific reagent binding, insufficient washing, or excessive detection development can create background (standard IHC practice). | Review blocking, washes, antibody concentration, and chromogen timing; compare a detection control with the stained slide (standard IHC practice). |
| An HPA-undetected cell type stains strongly. | Cross-reactivity or endogenous detection activity is possible (standard IHC practice); the discordance is clearest when cell identity is secure (HPA: negative tissue entries). | Verify the cell type, examine a control without primary antibody, and repeat with an independently validated antibody if available (standard IHC practice). |
| The dominant result is a membrane rim or broad nuclear staining. | That distribution conflicts with the reported cytoplasmic tissue pattern and SKA3's lack of a transmembrane segment (HPA: tissue IHC profile; UniProt Q8IX90: topology). | Check morphology, controls, and antibody identity before scoring; distinguish a mitotic kinetochore-associated focus from staining across ordinary nuclei (UniProt Q8IX90: mitotic localization; standard IHC practice). |
| Bone marrow is blank despite an expectation from RNA data. | HPA reports tissue-enhanced bone-marrow RNA but Not detected staining in hematopoietic cells (HPA: RNA specificity; tissue IHC). | Use the observed IHC result when choosing a staining reference; check testicular pachytene spermatocytes or epididymal glandular cells and document the RNA–IHC discrepancy (HPA: tissue IHC profile; reliability description). |
| A bright IF/ICC spindle result is used to justify a tissue-IHC call. | The subcellular locations are marked uncertain, and HPA lists validation separately by application (HPA: subcellular locations; antibody validation). | Interpret paraffin IHC against the reported tissue-cell pattern and its reliability caveat; evaluate IF/ICC localization on its own application-specific evidence (HPA: tissue IHC profile; reliability description; subcellular summary). |
Experiment evidence
IHC & IF images showing SKA3 staining
A07561-1 · IHCIHC analysis of SKA3 using anti-SKA3 antibody (A07561-1). SKA3 was detected in a paraffin-embedded section of human testis tissue. Heat medi
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000165480-SKA3/tissue/cerebral+cortex#imid_10815671
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000165480-SKA3/tissue/colon#imid_10815589
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000165480-SKA3/tissue/liver#imid_10815590
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000165480-SKA3/tissue/kidney#imid_10815583
HPA · ICC-IF HELHuman Protein Atlas immunofluorescence, HEL, antibody HPA039356; green = SKA3, blue = nucleus, red = microtubules.
HPA · ICC-IF HELHuman Protein Atlas immunofluorescence, HEL, antibody HPA039356; green = SKA3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for SKA3 IHC & IF
🧪Run testis first and assess staining in pachytene spermatocytes (HPA: High in testis pachytene spermatocytes). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes); other cells on the testis slide should show only background unless independently verified as SKA3-positive (HPA: testis row identifies pachytene spermatocytes only).
Positive control tissue: Epididymis (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show SKA3 in HEL, OE19, U2OS, Rh30, RPTEC/TERT1, with annotated localisation: Mitotic spindle (uncertain), Centrosome (uncertain) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a rabbit IgG isotype control matched to the primary antibody’s clonality where known (selected-SKU caption: rabbit anti-SKA3); a validated SKA3 knockout specimen provides a biological negative. For HRP–DAB staining, quench endogenous peroxidase and assess residual testis background on the no-primary slide (selected-SKU caption: HRP–DAB detection in testis).
⚠️Feasibility: Paraffin-section IHC in testis is documented with heat retrieval in EDTA at pH 8.0, but retrieval dependence has not been established (selected-SKU caption: testis IHC and EDTA retrieval). The caption does not report a fixative, and the supplied evidence reports no target-specific fixation window or fixation effect (selected-SKU caption: fixative unreported). It does not establish whether frozen sections or IF/ICC are easier; in testis, distinguish signal in the named spermatocyte population from surrounding background during scoring (HPA: High in pachytene spermatocytes).
HPA tissue IHC evidence for SKA3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low consistency between antibody staining and RNA expression data. Pending external verification.). 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 |
|---|---|---|---|---|
| Epididymis | Glandular cells | High | Protein (IHC) | HPA → |
| Testis | Pachytene spermatocytes | High | 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 → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced SKA3 IHC Tips
These questions focus on interpreting and troubleshooting SKA3 staining in paraffin sections, with one entry for companion IF experiments.
What retrieval conditions should I try when SKA3 staining is weak in paraffin sections?
Start with heat-mediated EDTA pH 8.0 retrieval for paraffin sections stained with A07561-1 (datasheet A07561-1). Keep section thickness, heating, cooling, and detection conditions consistent while comparing retrieval runs (standard IHC practice). The product image used this retrieval before incubation with 2 μg/ml primary antibody overnight at 4°C (datasheet A07561-1). If staining remains weak, vary retrieval duration cautiously and compare signal with tissue preservation on adjacent sections (standard IHC practice). Include a testis section as a reference, while remembering that an HPA staining pattern does not establish retrieval sensitivity for this antibody (datasheet A07561-1; HPA: Approved, pending external verification).
How can I assess whether fixation is causing weak SKA3 staining?
Target-specific SKA3 fixation sensitivity is unknown from the supplied evidence; the product image identifies a paraffin section but does not state its fixative (datasheet A07561-1). Record fixative, fixation duration, tissue thickness, and processing history before comparing sections (standard IHC practice). Stain adjacent sections in the same run using EDTA pH 8.0 retrieval and the same antibody concentration (datasheet A07561-1; standard IHC practice). Check whether tissue morphology and staining of an appropriate independent control also deteriorate, which would support a broader processing problem (standard IHC practice). Avoid attributing weak SKA3 signal to fixation alone without a controlled comparison (standard IHC practice).
Should I score cytoplasmic SKA3 staining or look only for mitotic structures?
Score the compartment and cell type separately: HPA reports cytoplasmic expression in testis and epididymis (HPA: tissue IHC profile). In mitotic cells, SKA3 localizes to the outer kinetochore and spindle microtubules in an NDC80 complex-dependent manner (UniProt Q8IX90). HPA subcellular imaging also lists centrosome, mitotic spindle, and cytosol locations, each marked uncertain (HPA: subcellular). Examine nuclear counterstain and mitotic morphology before calling a small punctum kinetochore-associated (standard IHC practice). Diffuse cytoplasmic staining can be recorded as a separate pattern, while compartment alone should not establish antibody specificity (HPA: tissue IHC profile; standard IHC practice).
Could isoforms or phosphorylation explain different SKA3 staining patterns?
SKA3 has 3 annotated isoforms and multiple modified residues, including phosphoserines at positions 34, 119, 139, 152, and 155 (UniProt Q8IX90). The supplied product caption does not identify the recognized epitope or establish isoform preference (datasheet A07561-1). Therefore, do not assign staining differences to a particular splice form or phosphorylation state without independent epitope information and controls (standard IHC practice). Compare serial sections under identical retrieval and detection conditions before interpreting a changed pattern biologically (standard IHC practice). If epitope information becomes available, check whether it spans a variable region or a modified residue before selecting additional controls (UniProt Q8IX90; standard IHC practice).
How should I use IF to investigate an ambiguous SKA3 IHC pattern?
Use IF on companion samples to examine whether SKA3 signal aligns with the expected cell type and mitotic structures (UniProt Q8IX90; standard IF practice). Multiplex with a validated marker for the cell population being assessed, and compare its distribution with SKA3 rather than assuming every positive cell is mitotic (standard IF practice). Select a fluorophore channel with low tissue autofluorescence and include unstained and single-label controls (standard IF practice). Because SKA3 has no transmembrane segment and localizes to intracellular structures, assess permeabilisation for access to intracellular epitopes (UniProt Q8IX90; standard IF practice). Verify that any IF pattern agrees with morphology; the supplied paraffin-section caption does not specify IF conditions (datasheet A07561-1).
What should I check when DAB obscures the SKA3 signal?
The documented paraffin-section workflow uses a 10% goat-serum block, 2 μg/ml primary antibody overnight at 4°C, and DAB detection (datasheet A07561-1). Run a no-primary control to assess secondary-reagent signal and include a peroxidase block to assess endogenous enzyme activity (standard chromogenic IHC practice). If background persists, inspect incomplete washing, excessive detection development, and staining concentrated at tissue edges (standard IHC practice). Reduce development or adjust blocking and washes one variable at a time while preserving a reference section in the run (standard IHC practice). Judge improvement by clearer cellular localization and maintained reference staining, not by reduced darkness alone (standard IHC practice).
How can I quantify SKA3 IHC without confusing cell composition with expression? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored compartment and cell population before analysis, then report the percentage of positive cells and staining intensity, or calculate an H-score (standard IHC practice). For discrete mitotic staining, report positive-cell density per mm² alongside the number of evaluable cells or mitoses (standard IHC practice). Normalize comparisons to the same cell type, section area, and scoring threshold across samples (standard IHC practice). HPA reports high staining in epididymal glandular cells and testicular pachytene spermatocytes, so pooled tissue averages could reflect changing cell composition (HPA: tissue IHC). Keep retrieval, DAB development, counterstain, and image acquisition consistent before comparing scores (standard IHC practice).
How can I distinguish credible SKA3 staining from artefact?
A credible pattern should fit the sampled cell type and compartment: HPA reports high staining in epididymal glandular cells and testicular pachytene spermatocytes (HPA: tissue IHC). SKA3 can also localize to the outer kinetochore and spindle microtubules during mitosis (UniProt Q8IX90). Examine unexpected membrane staining against the absence of a transmembrane segment, and inspect edge-restricted or necrotic staining for artefact (UniProt Q8IX90; standard IHC practice). Check a no-primary control and peroxidase blocking when DAB signal is widespread (standard chromogenic IHC practice). Treat apparent positives cautiously because HPA rates its tissue staining Approved while reporting low consistency with RNA and pending external verification (HPA: reliability).
Boster reagents
Best SKA3 / Spindle and kinetochore-associated protein 3 IHC Antibodies
One human-reactive anti-SKA3 antibody has real IHC data from a paraffin-embedded human testis section (catalog; IHC image caption). No IF image is supplied (catalog).
Real IHC data
Anti-C13orf3/SKA3 Antibody ®
Cat # A07561-1
A07561-1 is the only SKU that will render; its applications include IHC, and its image shows staining in a paraffin-embedded human testis section (catalog; IHC image caption). The catalog lists human reactivity and an IHC concentration of 2–5 μg/ml for A07561-1 (catalog).
Which to pick: Choose A07561-1 for human paraffin-section IHC: it is a rabbit polyclonal antibody with IHC listed, and its own image caption documents staining in human testis after heat retrieval in EDTA at pH 8.0 (catalog; IHC image caption). The caption does not report the fixative (IHC image caption). IF/ICC and cross-species use have no supporting application or reactivity listing for A07561-1 (catalog).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8IX90 (SKA3_HUMAN, Spindle and kinetochore-associated protein 3).
- Human Protein Atlas. SKA3 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. SKA3 subcellular location (ICC-IF): Mainly localized to the centrosome and mitotic spindle. In addition localized to the cytosol..
- Human Protein Atlas. SKA3 antibody validation summary (3 antibodies).
- SKA3 is a prognostic biomarker and associated with immune infiltration in bladder cancer. Hereditas 2022 — PMC9092687.
- SKA3 Expression as a Prognostic Factor for Patients with Pancreatic Adenocarcinoma. International journal of molecular sciences 2024 — PMC11120893.
- SKA3 Promotes tumor growth by regulating CDK2/P53 phosphorylation in hepatocellular carcinoma. Cell death & disease 2019 — PMC6895034.
- Integrative Multi-Omics Analysis of Identified SKA3 as a Candidate Oncogene Correlates with Poor Prognosis and Immune Infiltration in Lung Adenocarcinoma. International journal of general medicine 2022 — PMC9078431.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15057823 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for SKA3 (Spindle and kinetochore-associated protein 3). Expected localisation Cytoplasmic staining in tissue (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A07561-1). Validated IHC antibody: A07561-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Epididymis (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human testis tissue; fixative not specified (datasheet A07561-1); 2. Fixation: Image fixative and duration unreported (datasheet A07561-1); 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 A07561-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A07561-1); 8. Primary antibody: Rabbit anti-SKA3, 2-5 μg/ml (datasheet A07561-1); 9. Primary incubation: Overnight at 4 °C (datasheet A07561-1). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Staining and RNA show low concordance (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope coverage unknown (UniProt); Regulation: Expression regulation unreported (UniProt). Evidence: UniProt Q8IX90, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the centrosome and mitotic spindle. In addition…, Datasheet A07561-1, PMC11120893, PMC6895034. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/SKA3/immunohistochemistry-ihc-guide.