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TNIK / TRAF2 and NCK-interacting protein kinase · IHC design guide
Design Immunohistochemistry for TNIK
Plan TNIK immunohistochemistry in paraffin sections using tissue staining and catalog antibody evidence. The guide covers controls, detection and interpretation of the predominantly cytoplasmic tissue pattern (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
TNIK 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 most tissues (HPA tissue IHC) | |
| Staining pattern | Strong cytoplasmic staining in colon glandular cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A03934-3) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep paraffin-section fixation consistent across specimens. (selected-SKU IHC image A03934-3) | |
| Caveat | Staining shows low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | Intensity varies by tissue (HPA tissue IHC) | |
| Isoform / epitope | 8 isoforms; verify epitope coverage (UniProt) |
Section 1
Recommended TNIK IHC & IF Protocols
The catalog antibody uses EDTA pH 8.0 heat retrieval (datasheet A03934-3). Three published TNIK IHC protocols cover gastric, thyroid and colorectal specimens (PMC5424097; PMC10272969; PMC4619995).
| Sample | Paraffin-embedded human cervix squamous cell carcinoma tissue; fixative not specified (datasheet A03934-3) |
| Fixation | Image fixative and duration unreported (datasheet A03934-3); 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 A03934-3); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A03934-3) |
| Primary antibody | Rabbit anti-TNIK, 2-5 μg/ml (datasheet A03934-3) |
| Primary incubation | Overnight at 4 °C (datasheet A03934-3) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A03934-3) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | TNIK-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet A03934-3). Use each published retrieval condition with its corresponding antibody protocol (PMC5424097; PMC10272969; PMC4619995).
Section 2
What Is the Expected TNIK Staining Pattern?
TNIK is a non-transmembrane protein found in the cytoplasm and nucleus (UniProt Q9UKE5 topology; UniProt Q9UKE5 subcellular location). In paraffin-section IHC, expect mainly cytoplasmic staining in glandular cells, especially in appendix, colon, duodenum, gallbladder, rectum and small intestine (HPA: tissue IHC). HPA rates its IHC evidence Approved but reports low consistency between antibody staining and RNA expression; interpret unexpected patterns cautiously (HPA: tissue IHC reliability).
| Strong cytoplasmic staining in intestinal or gallbladder glandular cells. | This matches the reported high glandular-cell staining in colon, duodenum, rectum, small intestine and gallbladder (HPA: tissue IHC). Judge the result by cell type and compartment as well as intensity; a dark chromogen deposit alone does not establish TNIK localization (standard IHC interpretation). |
| Predominantly extracellular deposits or sharply outlined cell membranes, with little intracellular signal. | Treat this as a pattern mismatch: TNIK has no transmembrane segment, and HPA describes mainly cytoplasmic tissue staining (UniProt Q9UKE5 topology; HPA: tissue IHC). Check the matched negative control and staining distribution before attributing the deposits to TNIK (standard IHC practice). |
| Strong staining in a cell population reported as undetected, such as adipocytes or skin fibroblasts. | This is discordant with those HPA observations (HPA: adipocytes and skin fibroblasts not detected). Consider nonspecific antibody binding or endogenous detection activity, then inspect controls. HPA's low antibody-staining/RNA consistency means a single discordant specimen is insufficient to prove cross-reactivity (HPA: tissue IHC reliability). |
| Pale, widespread chromogen obscures cell boundaries and appears in several compartments. | Diffuse background makes compartment and cell-type scoring unreliable (standard IHC interpretation). Compare a section processed without primary antibody, review blocking and washing, and optimize antibody concentration and detection conditions as general IHC workflow checks (standard IHC practice). |
| No convincing signal in appendix or colon glandular cells. | HPA reports high staining in these glandular cells, so this is a useful sensitivity check (HPA: tissue IHC). Confirm that the expected cells are present and that detection and counterstaining worked; then review the IHC-validated antibody's instructions. Tissue-level expectations do not guarantee every specimen will stain (HPA: low antibody-staining/RNA consistency). |
💡Expected TNIK appearanceCall a positive result when glandular cells show clear, mainly cytoplasmic chromogen, potentially strong in HPA high-staining tissues; isolated extracellular, membrane-like or control-positive deposits should prompt a false-positive check (HPA: tissue IHC; UniProt Q9UKE5 topology; standard IHC interpretation).
| Tissue and cell selection | HPA reports high glandular-cell staining in appendix, colon, duodenum, gallbladder, rectum and small intestine, but no detected staining in adipocytes or skin fibroblasts (HPA: tissue IHC). Select and score the named cell population within each section; an unlabeled whole-tissue average can hide a cell-specific pattern (standard IHC practice). |
| Subcellular interpretation | HPA describes mainly cytoplasmic staining across tissues, while UniProt places TNIK in the nucleus, cytoplasm and recycling endosome; its endosome and cytoskeletal association is reported upon RAP2A overexpression (HPA: tissue IHC; UniProt Q9UKE5 subcellular location). Nuclear staining can be biologically plausible, but nuclear-only tissue IHC needs control review before acceptance. |
| Topology and processing | UniProt lists no transmembrane segment, signal peptide or propeptide and records a chain spanning residues 1–1360 (UniProt Q9UKE5 topology and processing). This supports an intracellular interpretation; it does not identify the catalog antibody's epitope or establish how paraffin processing affects that epitope. |
| Isoforms and antibody evidence | UniProt lists eight TNIK isoforms (UniProt Q9UKE5 isoforms). Their presence alone cannot predict which forms the catalog antibody detects because its epitope is unspecified. HPA lists HPA012128 and HPA012297 as IHC Approved; that status should be read alongside HPA's stated low antibody-staining/RNA consistency (HPA: antibodies; HPA: tissue IHC reliability). |
| IF/ICC Q&A: where should signal appear? | For the separate IF/ICC application, HPA reports supported nucleoplasmic localization with additional supported cytosolic localization (HPA: subcellular ICC-IF). The IF/ICC distribution can therefore differ from HPA's mainly cytoplasmic tissue IHC profile; use the application-specific guide to interpret IF/ICC images (HPA: tissue IHC; HPA: subcellular ICC-IF). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected glandular cells are unstained. | The section may lack the intended cells, or an IHC workflow step may have failed; HPA reports high staining in several glandular-cell populations (HPA: tissue IHC; standard IHC practice). | Verify glandular morphology, counterstain and a positive-control section; check the IHC-validated antibody's instructions and detection reagents before interpreting the sample as negative (standard IHC practice). |
| Only nuclei stain in tissue IHC. | UniProt permits nuclear TNIK, and ICC-IF supports nucleoplasmic localization, but HPA describes mainly cytoplasmic tissue staining (UniProt Q9UKE5 subcellular location; HPA: subcellular ICC-IF; HPA: tissue IHC). | Record the nuclear pattern as discordant with the usual tissue profile; inspect a matched control and repeat on an HPA high-staining glandular tissue before assigning specificity (standard IHC practice). |
| Signal outlines membranes or pools outside cells. | That distribution conflicts with TNIK's lack of a transmembrane segment and HPA's mainly cytoplasmic IHC profile (UniProt Q9UKE5 topology; HPA: tissue IHC). | Check the primary-antibody omission control, tissue morphology and chromogen deposits; optimize washing or detection if the control also develops color (standard IHC practice). |
| Diffuse staining prevents compartment scoring. | Broad background can arise from nonspecific binding or excess detection signal (standard IHC practice). | Compare controls, then adjust blocking, washing, antibody concentration or detection exposure one variable at a time; score only when cell boundaries and compartment can be distinguished (standard IHC practice). |
| A reported undetected cell population stains strongly. | HPA reports no detection in adipocytes and skin fibroblasts, but also warns of low consistency between antibody staining and RNA expression (HPA: tissue IHC). Endogenous activity or nonspecific binding remains possible (standard IHC practice). | Confirm cell identity, inspect the omission control and compare an HPA high-staining glandular section processed alongside it; describe unresolved staining as discordant rather than specific TNIK signal (HPA: tissue IHC; standard IHC practice). |
| Staining varies across specimens or regions. | HPA reports cytoplasmic expression in most tissues with cell-dependent intensity, and its IHC reliability note limits confidence in strict RNA concordance (HPA: tissue IHC). | Score the same cell population and compartment across sections, document intensity and distribution, and include a shared reference section in the run to separate sample variation from workflow variation (standard IHC practice). |
Experiment evidence
IHC & IF images showing TNIK staining
A03934-3 · IHCIHC analysis of TNIK using anti-TNIK antibody (A03934-3). TNIK was detected in a paraffin-embedded section of human cervix squamous cell car
A03934-3 · IHCIHC analysis of TNIK using anti-TNIK antibody (A03934-3). TNIK was detected in a paraffin-embedded section of human cervix squamous cell car
A03934-3 · IFIF analysis of TNIK using anti-TNIK antibody (A03934-3). TNIK was detected in an immunocytochemical section of PC-3 cells. Enzyme antigen re
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000154310-TNIK/tissue/cerebral+cortex#imid_3341964
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000154310-TNIK/tissue/colon#imid_3341906
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000154310-TNIK/tissue/liver#imid_3341907
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000154310-TNIK/tissue/kidney#imid_3341908
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA012128; green = TNIK, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA012128; green = TNIK, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for TNIK IHC & IF
🧪Run colon first: glandular cells should stain (HPA: High in colon glandular cells). Use adipose tissue adipocytes as the negative comparison (HPA: Not detected in adipocytes); on the colon slide, assess non-glandular cells for background without assuming they are TNIK-negative (HPA: colon result specifies glandular cells).
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show TNIK in A-431, U-251MG, U2OS, NIH 3T3, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only), a concentration-matched rabbit IgG isotype control, and TNIK-knockout material as a biological specificity control (caption: rabbit anti-TNIK antibody; standard IHC practice). Quench endogenous peroxidase and check background in the colon section before interpreting DAB staining (caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: The selected A03934-3 paraffin-section caption uses heat retrieval in EDTA at pH 8.0, but reports no fixative; a TNIK-specific fixation window or fixation effect is unreported (selected-SKU caption). The supplied evidence does not establish whether frozen sections or IF are easier than retrieved paraffin IHC; TNIK has supported nucleoplasmic and cytosolic IF localization (HPA: subcellular localization). In colon, endogenous peroxidase can create misleading DAB signal, so interpret glandular staining against the quenched and no-primary controls (HPA: High in colon glandular cells; caption: HRP/DAB detection; standard IHC practice).
HPA tissue IHC evidence for TNIK
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low 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 | Glandular cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Rectum | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Ovary | Ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Skin | Fibroblasts | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced TNIK IHC Tips
Troubleshooting TNIK staining in paraffin sections requires attention to retrieval, compartment, cell identity, and the limits of antibody validation.
What retrieval conditions should I start with for weak TNIK staining in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A03934-3). The catalog image used this retrieval before applying 2 μg/ml antibody overnight at 4°C, so keep those conditions consistent during the initial comparison (datasheet A03934-3). If staining remains weak, compare a second retrieval condition on matched sections while holding antibody concentration, detection, and development time constant; document tissue integrity alongside signal. Use a known positive glandular-cell section, such as colon, to distinguish failed retrieval from a genuinely low-signal specimen (HPA: High in colon glandular cells).
How should I troubleshoot fixation when TNIK staining is inconsistent across paraffin blocks?
The selected paraffin-section caption does not report a fixative, so TNIK-specific sensitivity to fixation is unknown (datasheet A03934-3: fixative not stated). Record the fixative, fixation duration, processing schedule, and block age for each specimen before comparing staining across batches. Run sections from a known positive control block through the same EDTA pH 8.0 retrieval and detection workflow as the study samples (datasheet A03934-3: retrieval condition; HPA: High in colon glandular cells). If a discrepancy tracks with processing history, repeat the comparison on similarly handled sections and report the limitation without assigning a TNIK-specific fixation mechanism.
How should I judge nuclear and cytoplasmic TNIK staining in tissue sections?
Score nuclear and cytoplasmic staining separately: TNIK is annotated in both compartments, while HPA tissue IHC describes predominantly cytoplasmic expression (UniProt Q9UKE5: subcellular location; HPA: cytoplasmic expression in most tissues). HPA cell imaging additionally supports nucleoplasmic and cytosolic localisation, but those observations do not establish identical distributions in every paraffin section (HPA: supported subcellular locations). Compare the pattern with adjacent morphology and a known positive glandular-cell control, such as colon (HPA: High in colon glandular cells). A punctate or cytoskeletal pattern needs independent confirmation because the reported recycling-endosome and cytoskeletal association occurs upon RAP2A overexpression (UniProt Q9UKE5: subcellular location).
Could TNIK isoforms or phosphosites explain differing staining patterns?
TNIK has 8 annotated isoforms, a kinase domain at residues 25–289, and a CNH domain at 1047–1334 (UniProt Q9UKE5: isoforms and domains). Ask which sequence the catalog antibody recognizes before attributing differences between specimens to isoform expression; the supplied caption does not identify its epitope (datasheet A03934-3: caption). TNIK also has annotated phosphorylation sites, including Thr-187 and Ser-324, but their effect on this antibody’s staining is unestablished (UniProt Q9UKE5: modified residues; datasheet A03934-3: caption). Compare independent antibodies with documented, distinct epitopes on matched sections if isoform or modification dependence is central to the study.
How can I assess TNIK by multiplex IF alongside a cell-type marker?
Treat IF as a separate assay and first establish whether the antibody produces specific cellular staining under the proposed fixation and permeabilisation conditions; the supplied catalog example documents chromogenic paraffin-section IHC (datasheet A03934-3). Pair TNIK with a marker identifying the cells being evaluated, and include single-stain controls to check channel bleed-through. Select a fluorophore in a channel with low tissue autofluorescence, confirmed on an unstained section, before comparing TNIK intensity between cells. Because TNIK has no transmembrane segment and is annotated in nuclear and cytoplasmic compartments, optimise permeabilisation for intracellular access while checking that cell boundaries and the companion marker remain interpretable (UniProt Q9UKE5: topology and subcellular location).
What should I check when TNIK DAB staining appears diffuse or patchy?
Compare a no-primary control with the stained section to assess secondary-reagent, endogenous peroxidase, and substrate-related background before changing the TNIK antibody. Include a peroxidase block and control DAB development time as general chromogenic IHC steps; the catalog image used a peroxidase-conjugated secondary and DAB detection (datasheet A03934-3). If background rises with primary concentration, titrate around the reported 2 μg/ml condition while keeping EDTA pH 8.0 retrieval fixed (datasheet A03934-3). Evaluate staining in intact cells away from folds, cut edges, and necrotic areas, then compare its compartment pattern with the annotated nuclear and cytoplasmic locations (UniProt Q9UKE5: subcellular location).
How should I quantify TNIK staining across paraffin-section samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, since tissue IHC is reported chiefly as cytoplasmic whereas TNIK also has a nuclear annotation (HPA: cytoplasmic expression in most tissues; UniProt Q9UKE5: subcellular location). For chromogenic sections, record the percentage of positive cells and an H-score from intensity categories 0–3; report nuclear and cytoplasmic results separately. Normalise each score to the number of eligible, morphologically intact cells in the prespecified region, rather than total section area. Keep retrieval, antibody concentration, DAB development, imaging, and positivity thresholds consistent, and review the same positive control across staining runs (datasheet A03934-3: EDTA pH 8.0 and 2 μg/ml antibody).
How can I separate credible TNIK staining from artefact or misleading controls?
A credible result should appear within intact cells, show a reproducible nuclear or cytoplasmic pattern, and survive comparison with a no-primary control (UniProt Q9UKE5: subcellular location). Use colon glandular cells as a positive reference, while recognising that HPA reports low consistency between its antibody staining and RNA data (HPA: High in colon glandular cells; HPA: Approved, low consistency). Do not treat normal cervix glandular-cell negativity as a direct contradiction of the catalog image: that image shows cervix squamous cell carcinoma, a different cell population (HPA: not detected in cervix glandular cells; datasheet A03934-3: image tissue). Edge staining, necrotic deposits, and endogenous peroxidase signal require exclusion through morphology and controls before assigning them to TNIK.
Boster reagents
Best TNIK / TRAF2 and NCK-interacting protein kinase IHC Antibodies
A03934-3 has IHC data from human paraffin sections of cervix squamous cell carcinoma and placenta, plus IF data from PC-3 cells (catalog image captions). Human and mouse reactivity are listed (catalog: reactivity).
Real IHC data
Anti-TNIK Antibody ®
Cat # A03934-3
A03934-3 will render with its IHC figure from a human paraffin-embedded cervix squamous cell carcinoma section (A03934-3 IHC caption). The same SKU lists IHC and ICC/IF applications, with additional IHC data from human placenta and IF data from PC-3 cells (catalog: applications; A03934-3 image captions).
Which to pick: For tissue IHC, choose A03934-3: its IHC caption documents a paraffin section, EDTA retrieval at pH 8.0, and primary antibody at 2 μg/ml; the fixative is unreported (A03934-3 IHC caption). For IF/ICC, choose A03934-3 because its IF caption documents staining in PC-3 cells at 5 μg/ml (A03934-3 IF caption). For cross-species planning, A03934-3 lists human and mouse reactivity, although the supplied IHC tissue examples are human; its host is rabbit and clonality is unreported (catalog: reactivity and host; A03934-3 IHC captions; catalog: clone).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9UKE5 (TNIK_HUMAN, TRAF2 and NCK-interacting protein kinase).
- Human Protein Atlas. TNIK tissue IHC expression (reliability: Approved).
- Human Protein Atlas. TNIK subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the cytosol..
- Human Protein Atlas. TNIK antibody validation summary (2 antibodies).
- The essential role of TNIK gene amplification in gastric cancer growth. Oncogenesis 2014 — PMC5424097.
- Expression analysis of TRAF2‑ and NCK‑interacting protein kinase (TNIK) and phosphorylated TNIK in papillary thyroid carcinoma. Oncology letters 2023 — PMC10272969.
- Prognostic significance of Traf2- and Nck- interacting kinase (TNIK) in colorectal cancer. BMC cancer 2015 — PMC4619995.
- Mendelian randomization integrated with multi-omics analysis identifies TNIK as a key gene in gut microbiota-induced IBD development. Frontiers in immunology 2025 — PMC12669205.
- PubMed PMID:10521462 — UniProt-cited evidence.
- PubMed PMID:9628581 — UniProt-cited evidence.
- PubMed PMID:12168954 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for TNIK (TRAF2 and NCK-interacting protein kinase). Expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A03934-3). Validated IHC antibody: A03934-3 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human cervix squamous cell carcinoma tissue; fixative not specified (datasheet A03934-3); 2. Fixation: Image fixative and duration unreported (datasheet A03934-3); 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 A03934-3); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A03934-3); 8. Primary antibody: Rabbit anti-TNIK, 2-5 μg/ml (datasheet A03934-3); 9. Primary incubation: Overnight at 4 °C (datasheet A03934-3). Watch for: Fixation: Keep paraffin-section fixation consistent across specimens. (selected-SKU IHC image A03934-3); Caveat: Staining shows low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: 8 isoforms; verify epitope coverage (UniProt); Regulation: Intensity varies by tissue (HPA tissue IHC). Evidence: UniProt Q9UKE5, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A03934-3, PMC5424097, PMC10272969. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/TNIK/immunohistochemistry-ihc-guide.