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TTK / Dual specificity protein kinase TTK · IHC design guide
Design Immunohistochemistry for TTK
Plan paraffin-section TTK IHC with the catalog antibody at 0.5–1 μg/mL (datasheet: A01132-1). Compare cytoplasmic staining in lymph-node germinal center cells with the not-detected signal in heart cardiomyocytes, and score by cell type (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
TTK 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 proliferative cells (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic signal, most abundant in proliferative cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A01132-1) | |
| Positive control | Epididymis+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Staining has medium RNA concordance; retesting is pending (HPA tissue IHC) | |
| Regulation | Higher in proliferative cells (HPA tissue IHC) | |
| Isoform / epitope | 2 isoforms; verify epitope coverage (UniProt) |
Section 1
Recommended TTK IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: A01132-1) is followed by two published TTK IHC protocols (PMC8721040; PMC13163004).
| Sample | Paraffin-embedded human rectal cancer tissue; fixative not specified (datasheet A01132-1) |
| Fixation | Image fixative and duration unreported (datasheet A01132-1); 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 A01132-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A01132-1) |
| Primary antibody | Rabbit anti-TTK, 0.5-1μg/ml (datasheet A01132-1) |
| Primary incubation | Overnight at 4 °C (datasheet A01132-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A01132-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | TTK-positive staining in glandular cells of epididymis (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several different cell types, most abundant in highly proliferative cells. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet: A01132-1); the published pH 9.0 method used a different antibody (PMC13163004).
Section 2
What Is the Expected TTK Staining Pattern?
In paraffin-section IHC, expect mainly cytoplasmic TTK staining, most conspicuous in highly proliferative cells (HPA tissue IHC). HPA reports High staining in lymph-node and tonsil germinal-center cells and testis pachytene spermatocytes (HPA tissue IHC). TTK has no transmembrane segment (UniProt P33981 topology). Tissue-IHC reliability is Enhanced, with medium staining–RNA consistency and retesting pending (HPA tissue IHC).
| Cytoplasmic staining in germinal-center cells or pachytene spermatocytes, with weaker neighboring cells. | This fits HPA's High staining in those named cell populations and its broader cytoplasmic profile (HPA tissue IHC). Score the specified cells and their compartment; a positive tissue does not imply uniform staining across every cell in the section (HPA tissue IHC). |
| Signal appears only at cell borders or exclusively throughout nuclei in the expected positive cells. | That differs from the mainly cytoplasmic tissue-IHC profile (HPA tissue IHC). Review morphology and detection background before calling it TTK; approved nucleolar localization comes from ICC-IF and does not establish an exclusively nuclear IHC pattern (HPA subcellular ICC-IF). |
| Strong staining dominates adipocytes or cardiomyocytes while the expected positive cells are weak. | HPA reports TTK as not detected in adipocytes and cardiomyocytes (HPA tissue IHC). Check whether the signal follows the cell type or the detection chemistry; cross-reactivity or endogenous detection activity can produce misleading chromogenic staining (general IHC practice). |
| Brown signal coats many structures without clear cell boundaries or a distinct cytoplasmic pattern. | Diffuse staining is difficult to attribute to TTK when it obscures HPA's cell-specific cytoplasmic profile (HPA tissue IHC). Compare a no-primary control and inspect blocking, washing, and chromogen development for nonspecific or endogenous signal (general IHC practice). |
| No convincing signal appears in lymph-node germinal-center cells or testis pachytene spermatocytes. | These are HPA High reference populations, so a blank result calls for an assay check before a biological negative call (HPA tissue IHC). Confirm cell identity and section quality, then review the IHC-validated antibody and its established retrieval and detection conditions (general IHC practice). |
💡Expected TTK appearanceA convincing positive is stronger, chiefly cytoplasmic staining in HPA High cell populations; indiscriminate border, nuclear-only, or widespread brown staining warrants investigation (HPA tissue IHC; HPA subcellular ICC-IF; general IHC practice).
| Reference-cell selection and proliferation | Germinal-center cells and pachytene spermatocytes provide HPA High tissue-IHC references; bone-marrow hematopoietic cells are Medium (HPA tissue IHC). UniProt reports TTK in rapidly proliferating cell lines, consistent with prioritizing proliferative populations, while the HPA calls remain cell-type specific (UniProt P33981 tissue specificity; HPA tissue IHC). |
| Tissue-IHC evidence strength | HPA labels tissue-IHC reliability Enhanced but notes medium consistency with RNA and pending retesting (HPA tissue IHC). Its listed mouse monoclonal CAB013229 has Enhanced IHC validation; the two listed rabbit antibodies have ICC approval without an IHC status in this payload (HPA antibodies). |
| Compartment and topology | HPA tissue IHC describes cytoplasmic expression, and UniProt lists no transmembrane segment (HPA tissue IHC; UniProt P33981 topology). TTK's spindle-checkpoint function does not, by itself, establish visible kinetochore staining in paraffin sections (UniProt P33981 function; general IHC interpretation). |
| Isoforms and epitope coverage | UniProt lists 2 TTK isoforms and a protein-kinase domain at residues 525–791 (UniProt P33981). The supplied record gives no antibody epitope, so isoform coverage cannot be inferred; consult the IHC-validated antibody's documented target region when interpreting a discrepant result (general IHC practice). |
| IF/ICC Q: Should the IHC pattern match ICC-IF exactly? | A: HPA ICC-IF places TTK mainly in the cytosol and additionally in nucleoli, both approved locations (HPA subcellular ICC-IF). Tissue IHC reports a cytoplasmic profile (HPA tissue IHC). Use the tissue-IHC evidence to judge paraffin sections; the ICC-IF observation alone does not require visible nucleoli in IHC. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive cells are blank in an IHC run. | An assay or detection failure is possible when HPA High reference cells lack signal (HPA tissue IHC; general IHC practice). | Check a known-positive section, antibody application, retrieval, and chromogenic detection against the validated IHC workflow (general IHC practice). |
| Only a faint signal appears in bone marrow. | HPA rates hematopoietic cells Medium, below its High germinal-center and pachytene-spermatocyte examples (HPA tissue IHC). | Compare matched runs with an HPA High reference population before treating a weaker bone-marrow result as failure (HPA tissue IHC; general IHC practice). |
| Brown staining appears in adipocytes or cardiomyocytes. | HPA reports both cell populations as not detected; nonspecific antibody binding or endogenous detection activity is possible (HPA tissue IHC; general IHC practice). | Identify the stained cells on the counterstain and compare a no-primary control; review the detection blocking step (general IHC practice). |
| Signal is diffuse across the section. | Poorly resolved chromogen or background can conceal the cytoplasmic, cell-specific profile described by HPA (HPA tissue IHC; general IHC practice). | Inspect no-primary control, wash performance, blocking, and chromogen development; score only cells with interpretable morphology (general IHC practice). |
| Staining is exclusively membranous or nuclear. | That distribution conflicts with HPA's chiefly cytoplasmic tissue-IHC profile; ICC-IF additionally reports nucleoli, not an exclusively nuclear pattern (HPA tissue IHC; HPA subcellular ICC-IF). | Recheck cell boundaries, counterstain, and controls, then compare localization in an HPA High reference cell type (HPA tissue IHC; general IHC practice). |
| Two TTK antibodies give different staining patterns. | HPA lists 2 isoforms, but the supplied evidence gives no epitopes; antibody-dependent recognition remains unresolved (UniProt P33981 isoforms; HPA antibodies). | Compare each antibody's documented IHC validation and target region, and prioritize the pattern in HPA-defined cells; CAB013229 has Enhanced IHC status in the supplied list (HPA antibodies; HPA tissue IHC). |
Experiment evidence
IHC & IF images showing TTK staining
A01132-1 · IHCIHC analysis of TTK using anti-TTK antibody (A01132-1). TTK was detected in paraffin-embedded section of human rectal cancer tissue. Heat me
A01132-1 · IHCIHC analysis of TTK using anti-TTK antibody (A01132-1). TTK was detected in paraffin-embedded section of rat intestine tissue. Heat mediated
A01132-1 · IFIF analysis of TTK using anti-TTK antibody (A01132-1). TTK was detected in immunocytochemical section of A431 cells. Enzyme antigen retrieva
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000112742-TTK/tissue/cerebral+cortex#imid_3901520
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000112742-TTK/tissue/colon#imid_3901446
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000112742-TTK/tissue/liver#imid_3901447
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000112742-TTK/tissue/kidney#imid_3901448
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB010166; green = TTK, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB010166; green = TTK, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for TTK IHC & IF
🧪Run rectum first and expect staining in its glandular cells (HPA: High in rectal glandular cells). Use adipose tissue as the negative tissue, with adipocytes expected to lack detectable staining (HPA: Not detected in adipocytes); on the rectal slide, treat adjacent nonglandular cells as an internal background reference only after confirming they are unstained.
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 TTK in A-431, U-251MG, U2OS, RPTEC/TERT1, with annotated localisation: Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) slide, a host- and clonality-matched isotype control, and TTK-knockout material or a validated peptide-block control if available (standard IHC practice). For rectal tissue with biotin-based DAB detection, check endogenous peroxidase and biotin background (catalog antibody caption: biotinylated secondary and DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A01132-1 paraffin-section caption does not state the fixative (catalog antibody caption). That caption uses heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required; the supplied evidence does not show that frozen sections or IF/ICC are easier (catalog antibody caption). Interpret rectal staining alongside background controls because the caption uses biotin-based DAB detection (catalog antibody caption; standard IHC practice).
HPA tissue IHC evidence for TTK
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data. Pending retesting.). 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 → |
| Lymph node | Germinal center cells | High | Protein (IHC) | HPA → |
| Rectum | Glandular cells | High | Protein (IHC) | HPA → |
| Testis | Pachytene spermatocytes | High | Protein (IHC) | HPA → |
| Tonsil | Germinal center cells | 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 → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced TTK IHC Tips
Troubleshoot TTK staining in paraffin sections by checking retrieval, cellular localisation, controls, and scoring before interpreting the chromogenic signal.
Which retrieval condition should I try first for weak TTK staining in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for this catalog antibody (datasheet A01132-1). The selected paraffin-section image used that condition, followed by 10% goat serum blocking and 1 μg/ml primary antibody overnight at 4°C (caption A01132-1). If signal is weak, compare controlled changes in heating time while keeping buffer, section thickness, detection chemistry, and development time constant (standard IHC practice). Use a concurrently processed positive control, such as lymph node germinal center cells, to separate retrieval failure from low expression in the study tissue (HPA: High in lymph node germinal center cells). Excessive heating can damage morphology and complicate cellular scoring (standard IHC practice).
Could fixation explain weak or uneven TTK staining?
The selected image identifies a paraffin-embedded section but does not state its fixative, so target-specific fixation sensitivity is unknown (caption A01132-1). Record the actual fixative, fixation duration, tissue thickness, and processing history for each block before comparing TTK staining across cases (standard IHC practice). If staining varies within a specimen, assess morphology and compare similarly processed positive-control sections before changing the antibody concentration (standard IHC practice). Keep retrieval at EDTA pH 8.0 during that comparison because this is the documented condition for the catalog antibody (datasheet A01132-1). Neither the tissue staining profile nor TTK sequence features establish a fixation preference (HPA tissue IHC; UniProt P33981).
How should I evaluate cytoplasmic and nuclear TTK staining?
Expect cytoplasmic staining in several cell types, with stronger tissue expression in highly proliferative cells (HPA tissue IHC). Cell imaging places TTK mainly in the cytosol and additionally in nucleoli, while UniProt supplies no subcellular annotation (HPA subcellular; UniProt P33981). Score cytoplasmic and nucleolar signals separately, using the counterstain to identify nuclei and assess whether apparent nuclear color is confined to nucleoli (standard IHC practice; HPA subcellular). Because TTK has no annotated transmembrane segment, a sharply continuous membrane rim warrants scrutiny for staining artefact (UniProt P33981 topology; standard IHC practice). Confirm any unusual pattern with a matched control and an independent antibody where available (standard IHC practice).
Can epitope location or isoforms change the staining pattern?
TTK has 2 reported isoforms, and its protein kinase domain spans residues 525–791 (UniProt P33981). The supplied catalog image does not identify the antibody epitope, so the observed stain cannot establish which isoform the antibody detects (caption A01132-1; UniProt P33981). Before comparing cases or antibodies, obtain each reagent's immunogen or epitope information and check whether that sequence is shared by the isoforms (standard IHC practice). TTK also has annotated modified residues, including N-terminal phosphorylation sites, but their effect on this antibody's binding is unreported (UniProt P33981). Resolve discordant patterns with an independent epitope and appropriate positive and negative controls (standard IHC practice).
How can I check TTK localisation by multiplex immunofluorescence?
Treat IF/ICC as a separate validation exercise; the selected antibody evidence is a chromogenic paraffin-section image (caption A01132-1). For glandular cells, pair TTK with an epithelial marker and a nuclear counterstain so the signal can be assigned to cells rather than adjacent tissue (HPA: High in rectal glandular cells; standard IF practice). Choose fluorophores after inspecting tissue autofluorescence in unstained controls, favoring channels with clean separation from that background (standard IF practice). Because the expected cytosolic and additional nucleolar signals are intracellular, use a compatible permeabilisation step and verify that it preserves morphology (HPA subcellular; standard IF practice). Include single-label controls when assessing multiplex bleed-through (standard IF practice).
What should I check when DAB signal appears diffuse?
The selected image used biotinylated secondary antibody, a streptavidin–biotin complex, and DAB detection after 10% goat serum blocking (caption A01132-1). Compare a no-primary control with the stained section to identify signal from detection reagents or endogenous tissue activity (standard IHC practice). Include a peroxidase block in the chromogenic workflow, and consider endogenous biotin when interpreting signal from this detection system (standard IHC practice; caption A01132-1). Review wash stringency, primary concentration, and DAB development while retaining a positive-control section in each run (standard IHC practice). Diffuse staining that does not resolve into cells or expected compartments requires validation before scoring (HPA tissue IHC; HPA subcellular; standard IHC practice).
How should I quantify TTK staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring: HPA reports cytoplasmic expression in several cell types and high signal in rectal glandular cells and lymph node germinal center cells (HPA tissue IHC). Within comparable regions, report the percentage of positive target cells and an H-score based on intensity categories, or positive-cell density per mm² when cell abundance is the outcome (standard IHC practice). Normalize cell-based scores to the number of evaluable cells of that type, and area-based counts to viable tissue area rather than the entire slide (standard IHC practice). Exclude folds, necrosis, and section edges using predefined rules (standard IHC practice). Keep imaging, DAB development, and scoring thresholds consistent across batches (standard IHC practice).
When is TTK staining convincing rather than artefactual?
A convincing result identifies individual cells and matches the reported cytoplasmic pattern, with possible nucleolar signal, in an appropriate population (HPA tissue IHC; HPA subcellular). Compare with a concurrently stained positive control, such as tonsil germinal center cells, and a no-primary control (HPA: High in tonsil germinal center cells; standard IHC practice). Question isolated membrane rims, broad staining at section edges, signal over necrosis, or color reproduced by the no-primary control (UniProt P33981 topology; standard IHC practice). Consider endogenous peroxidase or biotin-dependent detection background when interpreting the selected DAB workflow (caption A01132-1; standard IHC practice). HPA rates tissue evidence Enhanced but notes medium staining–RNA consistency and pending retesting, so corroborate unexpected biology independently (HPA tissue IHC).
Boster reagents
Best TTK / Dual specificity protein kinase TTK IHC Antibodies
The IHC-validated antibody has paraffin-section images from human rectal cancer and rat intestine (catalog IHC captions), plus IF data from A431 cells (catalog IF caption).
Real IHC data
Anti-TTK/Mps1/TTK Antibody ®
Cat # A01132-1
A01132-1 will render with an IHC figure from a paraffin-embedded human rectal cancer section (catalog IHC caption). The same SKU also has a rat intestine IHC image and an A431-cell IF image (catalog IHC and IF captions).
Which to pick: Choose A01132-1 for paraffin-section IHC; its human rectal cancer caption documents EDTA retrieval at pH 8.0 and 1 μg/ml primary antibody, but does not report the fixative (catalog IHC caption). For IF/ICC, A01132-1 has an A431-cell IF image using 2 μg/ml primary antibody (catalog IF caption). For cross-species planning, A01132-1 lists human, mouse and rat reactivity (catalog reactivity); its supplied IHC images document human and rat tissue only (catalog IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P33981 (TTK_HUMAN, Dual specificity protein kinase TTK).
- Human Protein Atlas. TTK tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. TTK subcellular location (ICC-IF): Mainly localized to the cytosol. In addition localized to the nucleoli..
- Human Protein Atlas. TTK antibody validation summary (3 antibodies).
- TTK is a favorable prognostic biomarker for triple-negative breast cancer survival. Oncotarget 2016 — PMC5348432.
- Key role of dual specificity kinase TTK in proliferation and survival of pancreatic cancer cells. British journal of cancer 2014 — PMC4453723.
- Identification and Validation of Hub Genes Associated with Bladder Cancer by Integrated Bioinformatics and Experimental Assays. Frontiers in oncology 2021 — PMC8721040.
- NFYA-Mediated TTK Up-Regulation Drives Fast Cell Cycle Progression and Its Inhibition Leads to Mitotic Catastrophe in Triple Negative Breast Cancer. Cancers 2026 — PMC13163004.
- PubMed PMID:1639825 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:14574404 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for TTK (Dual specificity protein kinase TTK). Expected localisation Cytoplasmic staining in proliferative cells (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A01132-1). Validated IHC antibody: A01132-1 at 0.5-1μg/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 rectal cancer tissue; fixative not specified (datasheet A01132-1); 2. Fixation: Image fixative and duration unreported (datasheet A01132-1); 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 A01132-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A01132-1); 8. Primary antibody: Rabbit anti-TTK, 0.5-1μg/ml (datasheet A01132-1); 9. Primary incubation: Overnight at 4 °C (datasheet A01132-1). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Staining has medium RNA concordance; retesting is pending (HPA tissue IHC); Isoform / epitope: 2 isoforms; verify epitope coverage (UniProt); Regulation: Higher in proliferative cells (HPA tissue IHC). Evidence: UniProt P33981, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the cytosol. In addition localized to the…, Datasheet A01132-1, PMC8721040, PMC13163004. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/TTK/immunohistochemistry-ihc-guide.