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NEK9 / Serine/threonine-protein kinase Nek9 · IHC design guide
Design Immunohistochemistry for NEK9
Plan NEK9 paraffin-section IHC around cytoplasmic staining in a few tissues (HPA tissue IHC). This guide covers consistent fixation, the catalog antibody’s 2–5 μg/ml IHC range (datasheet A04660-1), chromogenic detection and cell-level scoring.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NEK9 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 | Tissue staining is cytoplasmic (HPA tissue IHC); nuclear localisation is reported (UniProt) | |
| Staining pattern | Cytoplasmic staining in a few tissues, including glandular cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A04660-1) | |
| Positive control | Adrenal gland+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 samples. (standard IHC practice; not target-specific) | |
| Caveat | Antibody staining has low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | Staining-intensity regulation is unreported (UniProt) | |
| Isoform / epitope | No isoforms, signal peptide or propeptide are listed (UniProt) |
Section 1
Recommended NEK9 IHC & IF Protocols
The catalog antibody protocol uses EDTA pH 8.0 retrieval (datasheet: A04660-1). The published gastric cancer IHC protocols below use citrate retrieval (PMC7797683; PMC10344862).
| Sample | Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A04660-1) |
| Fixation | Image fixative and duration unreported (datasheet A04660-1); 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 A04660-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A04660-1) |
| Primary antibody | Rabbit anti-NEK9, 2-5 μg/ml (datasheet A04660-1) |
| Primary incubation | Overnight at 4 °C (datasheet A04660-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A04660-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | NEK9-positive staining in glandular cells of adrenal gland (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in a few tissues. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 for the catalog antibody (datasheet: A04660-1); test citrate retrieval when adapting the published gastric cancer protocols (PMC7797683; PMC10344862).
Section 2
What Is the Expected NEK9 Staining Pattern?
NEK9 is cytoplasmic and nuclear and has no transmembrane segment (UniProt Q8TD19). In paraffin section IHC, expect chiefly cytoplasmic staining in selected cell populations, including glandular, follicle, decidual and lymphoid cells reported by HPA (HPA tissue IHC). HPA rates the tissue profile Approved but reports low consistency between antibody staining and RNA expression; interpret the pattern alongside controls (HPA tissue IHC).
| Cytoplasmic staining in adrenal, duodenal, fallopian tube, rectal or seminal vesicle glandular cells. | This matches HPA's medium staining in those specific cell populations (HPA tissue IHC). Score the identified cells and their cytoplasm; adjacent cells need not share the same intensity. |
| Predominantly membrane-rim staining, with little cytoplasmic signal. | A membrane-only pattern is unexpected because NEK9 has no transmembrane segment and is annotated in cytoplasm and nucleus (UniProt Q8TD19). Check morphology and staining controls before calling it NEK9. |
| Strong staining in adipocytes or bone marrow hematopoietic cells. | HPA reports these cells as not detected (HPA tissue IHC). Unexpected signal may reflect cross-reactivity or endogenous detection activity; inspect a no-primary control and confirm cell identity. |
| Color covers stroma, empty spaces or much of the section without clear cell boundaries. | Diffuse color is difficult to assign to NEK9-positive cells. Assess background with a no-primary control and compare with the cell-restricted cytoplasmic profile (HPA tissue IHC). |
| No staining in adrenal glandular cells despite an interpretable section. | HPA reports medium staining in these cells (HPA tissue IHC). A negative result warrants checking the IHC-validated antibody, detection controls and tissue preservation before interpreting biological absence. |
💡Expected NEK9 appearanceA convincing positive is cell-associated, chiefly cytoplasmic chromogenic staining of medium intensity in HPA-listed glandular or other positive cells; widespread acellular color or isolated membrane rims are suspect (HPA tissue IHC; UniProt Q8TD19 topology).
| Cell population and tissue choice | HPA reports medium staining in ovarian follicle cells, placental decidual cells and appendix lymphoid tissue, alongside selected glandular cells (HPA tissue IHC). Choose a documented cell population when judging a control section. |
| Low-level and negative comparators | Kidney tubule cells, lung macrophages and pancreatic endocrine cells are reported at low levels; adipocytes and bone marrow hematopoietic cells are not detected (HPA tissue IHC). Weak or absent staining must be judged in the named cells. |
| Protein location and processing | NEK9 is annotated in cytoplasm and nucleus, lacks a transmembrane segment and has no signal peptide or propeptide (UniProt Q8TD19). These annotations support intracellular interpretation; they do not establish antigen retrieval needs or fixation sensitivity. |
| Evidence and antibody status | The listed antibody, HPA001405, has IHC status Approved, while HPA describes low consistency with RNA expression (HPA antibody record; HPA tissue IHC). Approved should not be read as an Enhanced, independently reproduced tissue pattern. |
| RNA abundance versus slide staining | UniProt describes NEK9 as most abundant in heart, liver, kidney and testis, whereas HPA reports low staining in kidney tubule cells and low tissue RNA specificity (UniProt Q8TD19; HPA tissue IHC). Do not turn an abundance statement into an IHC intensity threshold. |
| IF/ICC Q: What pattern is established? | A: UniProt annotates cytoplasm and nucleus, but HPA supplies no ICC-IF images or main location for NEK9 (UniProt Q8TD19; HPA subcellular record). Treat an IF pattern as a hypothesis for its separate guide, not an IHC-derived validation. |
| Situation | Likely cause | Next action |
|---|---|---|
| A documented medium-positive cell population is blank. | The negative result conflicts with HPA's staining report for that population (HPA tissue IHC); the specific technical cause is undetermined. | Verify cell identity, antibody and detection controls, and section quality; review the antibody's IHC-P instructions before changing general workflow settings. |
| Only kidney tubules or lung macrophages show faint color. | HPA reports low staining in these cell populations (HPA tissue IHC); faint color alone cannot establish assay failure. | Compare with a documented medium-positive population and a no-primary control, then score cell-associated signal separately from background. |
| Adipocytes or marrow hematopoietic cells stain strongly. | These cells are reported as not detected (HPA tissue IHC); cross-reactivity or endogenous detection activity is possible. | Check a no-primary control for detection background and confirm the stained cell type before assigning a NEK9-positive result. |
| Brown color appears throughout the section. | Diffuse color obscures the cell-restricted cytoplasmic profile (HPA tissue IHC); nonspecific detection background is a general IHC possibility. | Inspect a no-primary control and the counterstain, then adjust blocking or detection conditions according to the assay instructions. |
| Staining appears exclusively at cell borders. | A membrane-only result conflicts with NEK9's cytoplasmic and nuclear annotation and lack of transmembrane segment (UniProt Q8TD19). | Recheck section morphology and control staining; require convincing intracellular signal before treating the border pattern as NEK9. |
| A nuclear signal appears alongside cytoplasmic staining. | Nuclear localization is annotated by UniProt, while HPA summarizes tissue IHC as cytoplasmic (UniProt Q8TD19; HPA tissue IHC). | Record nuclear and cytoplasmic staining separately, check controls, and avoid claiming that the nuclear IHC pattern is HPA-confirmed. |
Experiment evidence
IHC & IF images showing NEK9 staining
A04660-1 · IHCIHC analysis of NEK9 using anti-NEK9 antibody (A04660-1). NEK9 was detected in a paraffin-embedded section of human colon cancer tissue. Hea
A04660-1 · IHCIHC analysis of NEK9 using anti-NEK9 antibody (A04660-1). NEK9 was detected in a paraffin-embedded section of human colon tissue. Heat media
A04660-1 · IFIF analysis of NEK9 using anti-NEK9 antibody (A04660-1). NEK9 was detected in a paraffin-embedded section of human colon cancer tissue. Heat
P04660 · IHCImmunohistochemical analysis of paraffin-embedded Human brain. Antibody was diluted at 1:100(4° overnight). High-pressure and temperature Tr
P04660 · IFImmunofluorescence analysis of HeLa cells, using NEK9 (Phospho-Thr210) Antibody. The picture on the right is blocked with the phospho peptid
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000119638-NEK9/tissue/cerebral+cortex#imid_562944
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000119638-NEK9/tissue/colon#imid_562870
HPA · RectumHuman Protein Atlas IHC, Rectum. Source: https://www.proteinatlas.org/ENSG00000119638-NEK9/tissue/rectum#imid_562846
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000119638-NEK9/tissue/liver#imid_562879
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NEK9 IHC & IF
🧪Run adrenal gland first: glandular cells should stain for NEK9 (HPA: Medium in adrenal gland glandular cells). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes); on the adrenal slide, assess non-glandular stromal cells for background staining without assuming they are biologically NEK9-negative.
Positive control tissue: Adrenal gland (Glandular cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for NEK9; derive a cell-line control from the positive tissue's cell type (Glandular cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control and an isotype-matched rabbit IgG control, alongside NEK9-knockout tissue or a validated immunizing-peptide block as a biological specificity control (selected-SKU caption: rabbit primary antibody). For chromogenic staining, block endogenous peroxidase and inspect the adrenal slide for residual pigment or background before scoring (selected-SKU caption: HRP/DAB detection).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU tissue-IHC caption does not state the fixative (selected-SKU caption: fixative not stated). Heat retrieval with EDTA at pH 8.0 was used for a paraffin section, but the evidence does not establish that NEK9 staining depends on that retrieval method (selected-SKU caption: EDTA pH 8.0). Relative ease of frozen-section IHC or IF is unreported; no HPA ICC-IF image lines are supplied, so check any IF signal against autofluorescence controls (HPA subcellular: no ICC-IF image lines).
HPA tissue IHC evidence for NEK9
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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Lymphoid tissue | Medium | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Medium | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | Medium | Protein (IHC) | HPA → |
| Ovary | Follicle cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic 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 NEK9 IHC Tips
Use the catalog antibody’s paraffin-section IHC conditions as a starting point, then assess NEK9 staining by compartment, cell type, and controls.
Which retrieval condition should I try first for weak NEK9 staining in paraffin sections?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A04660-1). The catalog image used this condition before incubating the primary antibody at 2 μg/ml overnight at 4°C (datasheet A04660-1). If staining remains weak, compare a small retrieval time series while holding antibody concentration and detection conditions constant (standard IHC practice). Check that tissue morphology remains intact and that the no-primary control stays clear (standard IHC practice). Interpret any improvement alongside expected cytoplasmic staining, because NEK9 is annotated in both cytoplasm and nucleus (UniProt Q8TD19).
How should I troubleshoot NEK9 staining when the section’s fixation history is uncertain?
The paraffin-section caption does not state a fixative, so NEK9-specific sensitivity to fixation is unknown (datasheet A04660-1). Record the available fixation history and compare sections processed alike before changing retrieval or antibody concentration (standard IHC practice). On a test section, begin with EDTA at pH 8.0 and the documented primary concentration of 2 μg/ml (datasheet A04660-1). If signal varies across samples, assess preservation, tissue edges, and morphology before assigning a biological difference (standard IHC practice). Neither NEK9’s intracellular location nor its listed phosphorylation sites establish how fixation affects this epitope (UniProt Q8TD19).
What cellular pattern supports a credible NEK9 IHC result?
Assess cytoplasmic staining first, while recording nuclear staining separately: NEK9 is annotated in both compartments, and the HPA tissue profile describes cytoplasmic expression in a few tissues (UniProt Q8TD19; HPA tissue IHC). Compare staining within identified cell populations rather than treating every stained area as equivalent (standard IHC practice). The HPA reports medium staining in appendix lymphoid tissue and several glandular cell populations, but its staining has low consistency with RNA expression (HPA tissue IHC). Use a counterstain and matched controls to distinguish intracellular signal from pigment or extracellular deposit (standard IHC practice). A nuclear-only result warrants validation before biological interpretation (UniProt Q8TD19).
Could epitope choice explain discrepant NEK9 staining between antibodies?
Check each antibody’s stated immunogen or epitope and application validation before comparing their staining patterns (standard IHC practice). The supplied NEK9 record lists 0 isoforms and a kinase domain spanning residues 52–308; it does not locate this catalog antibody’s epitope (UniProt Q8TD19; datasheet A04660-1). NEK9 also has listed modified residues, including phosphorylation near its N terminus, but their effect on this antibody’s binding is unreported (UniProt Q8TD19; datasheet A04660-1). Compare antibodies on adjacent sections with matched retrieval and detection conditions (standard IHC practice). Treat discordant patterns as an antibody-validation issue until an independent control supports one interpretation (standard IHC practice).
How can I assess NEK9 by IF alongside a cell-type marker?
Treat IF as a separate assay: the supplied catalog evidence documents chromogenic paraffin-section IHC, while HPA lists no cell lines with NEK9 ICC/IF images (datasheet A04660-1; HPA subcellular). Pair NEK9 with a validated marker for the cell population under study, and choose spectrally distinct fluorophores after checking tissue autofluorescence (standard IF practice). NEK9 has no transmembrane segment and is annotated in cytoplasm and nucleus, so optimize permeabilisation for intracellular access while preserving morphology (UniProt Q8TD19; standard IF practice). Include single-stain and no-primary controls to assess bleed-through and background (standard IF practice). Do not transfer the catalog IHC retrieval or concentration directly into an IF claim (datasheet A04660-1).
What should I change when NEK9 DAB staining looks diffuse or widespread?
First inspect the no-primary control and verify peroxidase blocking, washing, and DAB development time (standard chromogenic IHC practice). The catalog example used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and a peroxidase-linked secondary for 30 minutes at 37°C (datasheet A04660-1). If background persists, titrate primary antibody and detection strength while retaining a reference section in each run (standard IHC practice). Separate granular pigment, tissue-edge deposit, and diffuse chromogen from cell-shaped intracellular staining using the counterstain (standard IHC practice). HPA’s reported low staining consistency with RNA calls for caution when judging weak widespread signal (HPA tissue IHC).
How should I score NEK9 IHC across samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because NEK9 is annotated in cytoplasm and nucleus (UniProt Q8TD19). For each population, record the percentage of positive cells and staining intensity, then calculate an H-score if intensity categories are reproducible (standard IHC practice). If the biological question concerns infiltrating positive cells, report their density per mm² of viable tissue and state the counted area (standard IHC practice). Normalize comparisons to the same cell population, compartment, section area, and staining run; exclude necrosis and damaged edges by a predefined rule (standard IHC practice). Report controls because HPA tissue staining shows low consistency with RNA expression (HPA tissue IHC).
When is apparent NEK9 positivity more likely to be artefact?
Interpret cell-shaped cytoplasmic staining as plausible, and assess nuclear signal separately, because NEK9 is annotated in both compartments (UniProt Q8TD19). Staining confined to extracellular material, necrotic regions, or section edges is suspect and should be checked against morphology and the no-primary control (standard IHC practice). Persistent DAB signal without primary antibody raises concern for endogenous peroxidase activity or detection background (standard chromogenic IHC practice). Check the stained cell type: HPA reports medium signal in appendix lymphoid tissue but no detection in bone-marrow hematopoietic cells, with low consistency between staining and RNA data (HPA tissue IHC). Confirm surprising patterns with an independent antibody or orthogonal assay before assigning NEK9-specific meaning (standard IHC practice).
Boster reagents
Best NEK9 / Serine/threonine-protein kinase Nek9 IHC Antibodies
Two NEK9 antibodies have IHC images from human paraffin sections; IF images cover human paraffin sections and HeLa cells (catalog image captions).
Real IHC data
Anti-NEK9 Antibody ®
Cat # A04660-1
Real IHC data
Anti-Phospho NEK9 (Thr210) Antibody
Cat # P04660
A04660-1 has IHC images from human colon, colon cancer, and breast cancer paraffin sections, plus IF images from human colon and breast cancer paraffin sections (A04660-1 image captions). P04660 targets phospho-NEK9 Thr210 and has an IHC image from human brain paraffin sections and an IF image from HeLa cells (P04660 title and image captions).
Which to pick: Choose A04660-1 for total NEK9 tissue IHC or tissue IF in human paraffin sections (A04660-1 applications, reactivity, and image captions). Choose P04660 for phospho-NEK9 Thr210 IHC or ICC/IF; it is listed as polyclonal and reactive with human, mouse, and rat, although its IHC image shows only human brain (P04660 title, dilution data, reactivity, and image captions). Both IHC captions describe paraffin sections and antigen retrieval; neither reports the fixative (A04660-1 and P04660 IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8TD19 (NEK9_HUMAN, Serine/threonine-protein kinase Nek9).
- Human Protein Atlas. NEK9 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. NEK9 subcellular location (ICC-IF): Highest expression in ASC52telo: 82.9 nTPM.
- Human Protein Atlas. NEK9 antibody validation summary (1 antibodies).
- Overexpression of the NEK9-EG5 axis is a novel metastatic marker in pathologic stage T3 colon cancer. Scientific reports 2023 — PMC9825400.
- NEK9 regulates primary cilia formation by acting as a selective autophagy adaptor for MYH9/myosin IIA. Nature communications 2021 — PMC8172835.
- NEK9, a novel effector of IL-6/STAT3, regulates metastasis of gastric cancer by targeting ARHGEF2 phosphorylation. Theranostics 2021 — PMC7797683.
- Cancer associated fibroblast derived SLIT2 drives gastric cancer cell metastasis by activating NEK9. Cell death & disease 2023 — PMC10344862.
- PubMed PMID:11864968 — UniProt-cited evidence.
- PubMed PMID:12101123 — UniProt-cited evidence.
- PubMed PMID:12056414 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NEK9 (Serine/threonine-protein kinase Nek9). Expected localisation Tissue staining is cytoplasmic (HPA tissue IHC); nuclear localisation is reported (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A04660-1). Validated IHC antibody: A04660-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A04660-1); 2. Fixation: Image fixative and duration unreported (datasheet A04660-1); 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 A04660-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A04660-1); 8. Primary antibody: Rabbit anti-NEK9, 2-5 μg/ml (datasheet A04660-1); 9. Primary incubation: Overnight at 4 °C (datasheet A04660-1). Watch for: Fixation: Keep paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific); Caveat: Antibody staining has low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: No isoforms, signal peptide or propeptide are listed (UniProt); Regulation: Staining-intensity regulation is unreported (UniProt). Evidence: UniProt Q8TD19, HPA tissue IHC · Approved, HPA cell-line RNA · Highest expression in ASC52telo: 82.9 nTPM, Datasheet A04660-1, PMC7797683, PMC10344862. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NEK9/immunohistochemistry-ihc-guide.