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CCNK / Cyclin-K · IHC design guide
Design Immunohistochemistry for CCNK
Plan chromogenic IHC for CCNK using its widespread nuclear tissue pattern (HPA tissue IHC). Compare staining across cell types, since some have no detectable signal (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CCNK 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 | Nuclear staining in tissue (HPA tissue IHC) | |
| Staining pattern | Widespread nuclear staining across cell types (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, heat-mediated (datasheet A06254) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Adipose tissue+2 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across samples. (standard IHC practice; not target-specific) | |
| Caveat | Some cell types have no detectable stain (HPA tissue IHC) | |
| Regulation | Staining intensity varies by cell type (HPA tissue IHC) | |
| Isoform / epitope | Four isoforms; epitope coverage is unknown (UniProt) |
Section 1
Recommended CCNK IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet A06254) with a published CCNK tissue IHC protocol (PMC11898804).
| Sample | Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A06254) |
| Fixation | Image fixative and duration unreported (datasheet A06254); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6.0 (datasheet A06254); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-CCNK, 1:50-1:200 (datasheet A06254) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CCNK-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 heat retrieval for the catalog antibody (datasheet A06254). The published protocol used high-pH CC1 retrieval (PMC11898804).
Section 2
What Is the Expected CCNK Staining Pattern?
CCNK should appear predominantly in nuclei across many tissues: UniProt assigns it to the nucleus and reports no transmembrane segment (UniProt O75909: subcellular location and topology). HPA describes ubiquitous nuclear tissue staining, with high signal in selected glandular, glial, and trophoblastic cells (HPA: tissue IHC). Interpret that pattern with the reported medium consistency between staining and RNA data (HPA: Supported reliability).
| Nuclear chromogen in adrenal or parathyroid glandular cells, cortical glial cells, or placental trophoblastic cells (HPA: High in each). | This fits the reported tissue IHC pattern (HPA: ubiquitous nuclear expression). Compare nuclei within the expected cell population and their neighbors; a positive call should reflect nuclear staining, rather than color spread across the section (UniProt O75909: nucleus; standard IHC practice). |
| Strong cytoplasmic or membranous staining with little nuclear signal. | That distribution conflicts with the annotated nuclear location and lack of a transmembrane segment (UniProt O75909: location and topology). Review morphology, counterstain, and the no-primary control before attributing the color to CCNK (standard IHC practice). |
| Prominent staining in adipocytes, liver cholangiocytes, or splenic red-pulp cells. | These specific cell populations were reported as not detected (HPA: tissue IHC). Check whether color follows nuclei and persists without primary antibody; cross-reactivity or endogenous detection activity is possible (standard IHC practice). A different cell population in the same tissue needs its own assessment. |
| Diffuse color over nuclei and extracellular areas, with poor cellular boundaries. | This is hard to score as nuclear CCNK (UniProt O75909: nucleus). Uneven reagent coverage, inadequate washing, or endogenous detection activity can raise background (standard IHC practice). Compare the no-primary section and adjust the general detection workflow before interpreting intensity. |
| No nuclear staining in adrenal or parathyroid glandular cells, or in placental trophoblastic cells. | Those populations have high reported staining and can serve as reference positives (HPA: High in each). An absent result may reflect a failed stain or unsuitable assay conditions; verify that the IHC antibody, detection reagents, and control section work before calling the specimen negative (standard IHC practice). |
💡Expected CCNK appearanceCall CCNK positive when chromogen is predominantly nuclear in the relevant cells, with strong signal plausible in the HPA high populations; dominant cytoplasmic color or staining of HPA not-detected cell populations warrants control review (UniProt O75909: nucleus; HPA: tissue IHC; standard IHC practice).
| Tissue and cell population | Expected intensity differs by cell type: HPA reports High in adrenal and parathyroid glandular cells, cortical glia, and placental trophoblasts, but Low in alveolar cells and several intestinal glandular populations (HPA: tissue IHC). Score the identified cells, rather than assigning one intensity to an entire organ. |
| Evidence and antibody choice | The tissue pattern has Supported reliability with medium staining–RNA consistency (HPA: tissue IHC). HPA077073 has Supported IHC validation; HPA000645 has no IHC status in this record (HPA: antibody validation). An ICC result alone does not establish paraffin-section performance. |
| Isoforms and epitope coverage | UniProt lists 4 CCNK isoforms (UniProt O75909: isoforms). The supplied record gives no antibody epitope or isoform coverage, so it cannot predict whether a particular isoform stains differently. Check the chosen IHC antibody's documented epitope before interpreting an unexpected distribution. |
| Processing and topology | CCNK is annotated as a 1–580 chain, with no signal peptide, propeptide, or transmembrane segment (UniProt O75909: processing and topology). Those annotations support a nuclear expectation, but they provide no basis for predicting antigen-retrieval requirements or fixation sensitivity. |
| IF/ICC comparison | HPA reports enhanced nucleoplasmic localization by ICC-IF and lists MCF-7, U-251MG, SiHa, and U2OS images (HPA: subcellular; antibody validation). This answers the IF localization question; it does not supply an IF protocol or replace the tissue IHC pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| Positive-control nuclei are blank. | A failed stain or unsuitable assay condition is possible when HPA high populations show no nuclear color (HPA: tissue IHC; standard IHC practice). | Repeat with a documented IHC antibody and an HPA high cell population; verify retrieval, primary incubation, detection, and counterstain against the assay's established settings (standard IHC practice). No CCNK-specific retrieval condition is supplied. |
| Nuclei are weak in the test tissue. | Low signal may be expected in alveolar cells or several intestinal glandular populations (HPA: Low in these cells). A weak section alone cannot distinguish biology from assay performance. | Check a matched high-staining control, then score the identified cells at the same detection settings (HPA: High in selected populations; standard IHC practice). Avoid increasing signal until background is assessed. |
| Cytoplasm stains more strongly than nuclei. | The pattern conflicts with nuclear CCNK and nucleoplasmic ICC-IF localization (UniProt O75909: nucleus; HPA: nucleoplasm). Background or nonspecific binding is possible (standard IHC practice). | Compare an adjacent no-primary section, inspect cell boundaries and nuclear counterstain, and confirm the antibody's IHC validation (standard IHC practice; HPA: HPA077073 IHC Supported). |
| Adipocytes, cholangiocytes, or splenic red-pulp cells appear strongly positive. | Those cell populations were not detected in the HPA tissue profile (HPA: tissue IHC); cross-reactivity or endogenous detection activity is possible (standard IHC practice). | Identify the stained cell type on the counterstained section and inspect no-primary and detection controls; address endogenous activity or nonspecific staining before assigning CCNK positivity (standard IHC practice). |
| Color is diffuse across the whole section. | Poor separation of nuclear signal from background prevents a confident localization call (UniProt O75909: nucleus; standard IHC practice). Inadequate washing or excess detection background may contribute. | Compare the no-primary section, check reagent coverage and wash steps, and reassess nuclear staining only after background is controlled (standard IHC practice). |
| IF/ICC appears nucleoplasmic, but paraffin IHC disagrees. | HPA's enhanced nucleoplasmic ICC-IF finding and its tissue IHC profile are distinct observations (HPA: subcellular and tissue IHC). The supplied evidence does not identify a CCNK-specific fixation or retrieval cause. | Judge the paraffin result against IHC controls and the expected cell populations (HPA: tissue IHC; standard IHC practice). Use the separate IF/ICC guide for that assay's workflow. |
Experiment evidence
IHC & IF images showing CCNK staining
A06254 · IHCImmunohistochemistry of paraffin-embedded human breast cancer using Cyclin K Rabbit pAb at dilution of 1:100 .Perform high pressure antigen
A06254 · IHCImmunohistochemistry of paraffin-embedded mouse spinal cord using Cyclin K Rabbit pAb at dilution of 1:100 .Perform high pressure antigen re
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000090061-CCNK/tissue/cerebral+cortex#imid_20375525
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000090061-CCNK/tissue/colon#imid_20376667
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000090061-CCNK/tissue/liver#imid_20376660
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000090061-CCNK/tissue/kidney#imid_20376669
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA000645; green = CCNK, blue = nucleus, red = microtubules.
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA000645; green = CCNK, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CCNK IHC & IF
🧪Run adrenal gland first and look for staining in its glandular cells (High; HPA: adrenal gland, glandular cells). Use adipose tissue as the negative comparison (HPA: adipocytes, Not detected); on the adrenal slide, compare staining with adjacent nonglandular cells as a background reference, without assuming those cells are CCNK-negative.
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CCNK in MCF-7, U-251MG, SiHa, U2OS, with annotated localisation: Nucleoplasm (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a matched rabbit polyclonal isotype control for the captioned antibody (caption: Rabbit pAb), and CCNK knockout material as a biological negative if available. For chromogenic IHC, quench endogenous peroxidase and inspect the adrenal section for pigment that could be mistaken for staining (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence, and the selected SKU caption does not state a fixative (caption: A06254). The paraffin-section example uses high-pressure retrieval in 10 mM citrate, pH 6.0, but does not establish that CCNK staining depends on that retrieval method (caption: A06254). HPA shows nucleoplasmic ICC-IF localisation, but the supplied evidence does not establish that frozen sections or IF are easier than paraffin IHC (HPA: Nucleoplasm, enhanced); inspect adrenal pigment when interpreting chromogenic staining (standard IHC practice).
HPA tissue IHC evidence for CCNK
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Glial cells | High | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | High | Protein (IHC) | HPA → |
| Parathyroid gland | Glandular cells | High | Protein (IHC) | HPA → |
| Placenta | Trophoblastic cells | High | Protein (IHC) | HPA → |
Section 3
Advanced CCNK IHC Tips
Troubleshoot CCNK staining by checking nuclear localisation, retrieval conditions and assay controls (UniProt O75909 localization; datasheet A06254).
How should I recover CCNK staining in paraffin sections without increasing background?
Start with heat-mediated retrieval in 10 mM citrate buffer, pH 6.0, using the high-pressure method documented for the catalog antibody (datasheet A06254). The pictured paraffin-embedded breast cancer section used this retrieval and antibody at 1:100, but its fixative was not reported (caption A06254). If nuclear staining is weak, compare a modestly longer heating interval on serial sections while keeping cooling, wash steps, and detection constant (standard IHC practice). Judge improvement by nuclear signal relative to matched no-primary background, because CCNK is nuclear and the HPA pattern is predominantly nuclear (UniProt O75909 localization; HPA tissue IHC).
What fixation conditions should I use when nuclear CCNK signal is inconsistent?
The selected paraffin-section caption does not report a fixative, so target-specific CCNK sensitivity to fixation duration or chemistry is unknown (caption A06254). For a controlled troubleshooting series, record fixative, tissue thickness, delay before fixation, and fixation time, then vary one factor across matched sections (standard IHC practice). Keep the 10 mM citrate, pH 6.0 retrieval and the catalog antibody's 1:100 pictured dilution constant during that comparison (datasheet A06254; caption A06254). Evaluate nuclear staining and tissue preservation together, using the same counterstain and detection batch, and avoid assigning any observed difference to CCNK biology without independent validation (standard IHC practice).
Where should CCNK staining appear, and how should I handle cytoplasmic signal?
Score CCNK chiefly in nuclei: UniProt assigns it to the nucleus, and HPA reports enhanced nucleoplasmic localization and ubiquitous nuclear tissue staining (UniProt O75909 localization; HPA subcellular; HPA tissue IHC). CCNK has no annotated transmembrane segment or signal peptide, so a crisp membrane outline is not its expected pattern (UniProt O75909 topology and processing). Examine nuclear signal at matched magnification with a counterstain and record cytoplasmic staining separately rather than pooling compartments (standard IHC practice). If cytoplasm dominates, compare no-primary and secondary-only controls, assess section edges, and repeat staining with adjusted antibody concentration or detection exposure before interpreting it as CCNK (standard IHC practice).
Can this IHC antibody distinguish CCNK isoforms or phosphorylation states?
UniProt lists four CCNK isoforms and phosphoserines at residues 324, 328, 329, and 340 (UniProt O75909 isoforms and modified residues). The supplied caption identifies a rabbit polyclonal antibody but gives no immunogen, mapped epitope, or isoform-specific validation, so isoform and phosphorylation-state discrimination is unestablished (caption A06254). Compare staining with an independently validated antibody whose epitope is known, or with orthogonal molecular measurements, before assigning a tissue pattern to one isoform (standard IHC validation practice). Keep retrieval and detection conditions matched across sections, since changes in epitope accessibility can alter apparent staining without demonstrating a biological isoform shift (standard IHC practice).
How can I confirm nuclear CCNK by multiplex IF in tissue?
Treat multiplex IF as a separate optimization because the supplied product image documents chromogenic staining of paraffin sections, without a stated fixative or IF protocol (caption A06254). Pair CCNK with a validated marker for the cell population being scored and a nuclear counterstain, then examine colocalisation within individual nuclei (standard IF practice; HPA subcellular: nucleoplasm). Choose a fluorophore channel with low tissue autofluorescence, and include unstained, single-color, and no-primary controls to set thresholds and check bleed-through (standard IF practice). Because CCNK is nuclear and has no transmembrane segment, optimize permeabilisation for nuclear access after fixing the IF sample, while preserving morphology (UniProt O75909 localization and topology; standard IF practice).
How do I separate weak nuclear CCNK staining from nonspecific chromogen?
Use serial sections with no primary antibody and a matched negative reagent control to measure detection-system background before changing the CCNK antibody dilution (standard IHC practice). In chromogenic work, quench endogenous peroxidase before the detection reagent and inspect pigmented or damaged areas before interpreting DAB deposits (standard IHC practice). The pictured catalog antibody was used at 1:100 after 10 mM citrate, pH 6.0 high-pressure retrieval, which provides a starting condition, not a tissue-wide background threshold (caption A06254). Prefer crisp nuclear labeling over diffuse stromal, cytoplasmic, or edge staining, consistent with reported nuclear and nucleoplasmic CCNK localization (UniProt O75909 localization; HPA subcellular).
What scoring method best captures CCNK IHC across heterogeneous sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the analyzed compartment as nuclei and score intact cells in prespecified regions, because CCNK is reported as nuclear and HPA describes ubiquitous nuclear expression (UniProt O75909 localization; HPA tissue IHC). Record the percentage of positive nuclei and intensity categories, then calculate an H-score as 1×% weak + 2×% moderate + 3×% strong on a 0–300 scale (standard IHC scoring practice). Normalize counts to evaluable nuclei in each annotated cell population, or report positive nuclei per mm² with the sampled area stated (standard quantitative pathology practice). Keep thresholds, illumination, counterstain, and acquisition settings fixed across batches, and exclude necrotic or folded tissue before comparison (standard IHC practice).
Which CCNK IHC patterns support a true positive rather than artefact?
A convincing positive is reproducible nuclear staining in intact cells, aligning with UniProt nuclear localization and HPA's nucleoplasmic and tissue nuclear patterns (UniProt O75909 localization; HPA subcellular; HPA tissue IHC). HPA reports high staining in adrenal glandular cells and placental trophoblastic cells, whereas adipocytes were not detected; these are contextual comparators, not absolute controls (HPA tissue IHC). Strong membrane outlines, section-edge gradients, necrotic deposits, or signal persisting without primary antibody warrant an artefact check, including endogenous peroxidase activity in chromogenic detection (standard IHC practice; UniProt O75909 topology). Interpret discordant regions cautiously because HPA rates tissue evidence as supported with medium RNA-staining consistency (HPA tissue IHC).
Boster reagents
Best CCNK / Cyclin-K IHC Antibodies
A06254 has real IHC images from paraffin-embedded human breast cancer and mouse spinal cord (catalog image captions); rat reactivity is listed without a tissue image (catalog reactivity); no IF data are supplied (catalog).
Real IHC data
Anti-Cyclin-K CCNK Antibody
Cat # A06254
A06254 is listed for IHC and has images of paraffin-embedded human breast cancer and mouse spinal cord, each stained at 1:100 after citrate pH 6.0 retrieval (catalog applications; catalog image captions). Human, mouse and rat reactivity is listed, but the supplied images document human and mouse tissue only (catalog reactivity; catalog image captions).
Which to pick: Choose A06254 for paraffin-section IHC using its stated 1:50–1:200 range; its images document paraffin sections, but do not report the fixative (datasheet: IHC dilution; catalog image captions). There is no IF/ICC choice supported by this catalog: A06254 lists IHC and WB, with no IF dilution or image (catalog applications; catalog IF fields). A06254 is the only listed cross-species option for human, mouse and rat, though tissue images are supplied only for human and mouse (catalog reactivity; catalog image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O75909 (CCNK_HUMAN, Cyclin-K).
- Human Protein Atlas. CCNK tissue IHC expression (reliability: Supported).
- Human Protein Atlas. CCNK subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. CCNK antibody validation summary (2 antibodies).
- Prognostic Evaluation and Functional Characterization of Cyclin K Expression in Endometrial Cancer: Immunohistochemical and In Silico Analysis. Cancers 2025 — PMC11898804.
- PubMed PMID:9632813 — UniProt-cited evidence.
- PubMed PMID:12508121 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CCNK (Cyclin-K). Expected localisation Nuclear staining in tissue (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, heat-mediated (datasheet A06254). Validated IHC antibody: A06254 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A06254); 2. Fixation: Image fixative and duration unreported (datasheet A06254); 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: Citrate pH 6.0 (datasheet A06254); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-CCNK, 1:50-1:200 (datasheet A06254); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across samples. (standard IHC practice; not target-specific); Caveat: Some cell types have no detectable stain (HPA tissue IHC); Isoform / epitope: Four isoforms; epitope coverage is unknown (UniProt); Regulation: Staining intensity varies by cell type (HPA tissue IHC). Evidence: UniProt O75909, HPA tissue IHC · Supported, HPA ICC-IF · nucleoplasm., Datasheet A06254, PMC11898804. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CCNK/immunohistochemistry-ihc-guide.