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CCNK / Cyclin-K · IHC design guide
Design Immunohistochemistry for CCNK
Plan CCNK IHC-P around its broad nuclear tissue pattern (HPA tissue IHC). Compare high staining in placental trophoblasts with undetected staining in adipocytes, and optimize the catalog antibody within 1:50–1:200 (HPA tissue IHC; datasheet A06254).
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 across tissues (HPA tissue IHC) | |
| Staining pattern | Broad nuclear staining in tissue cells (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 | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A06254) | |
| Caveat | Adipocytes may lack detectable staining (HPA tissue IHC) | |
| Regulation | Widely expressed; highest in testis (UniProt) | |
| Isoform / epitope | 4 isoforms; epitope effects unknown (UniProt) |
Section 1
Recommended CCNK IHC & IF Protocols
The catalog antibody has a datasheet IHC-P protocol (datasheet A06254). The published endometrial tissue protocol below uses a different retrieval buffer (PMC11898804 methods).
| 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). Use high-pH CC1 for the published workflow (PMC11898804 methods).
Section 2
What Is the Expected CCNK Staining Pattern?
CCNK should appear predominantly in nuclei in paraffin-section IHC (UniProt O75909: nucleus; HPA: ubiquitous nuclear expression). Expect staining across many cell types, with high signal reported in adrenal, epididymal, and parathyroid glandular cells, cerebral cortical glia, and placental trophoblasts (HPA: tissue IHC). HPA rates the tissue pattern Supported, with medium consistency between antibody staining and RNA data (HPA: reliability). CCNK has no transmembrane segment (UniProt O75909: topology).
| Clear nuclear staining in adrenal glandular cells or placental trophoblasts, with nuclei identifiable by counterstain. | This matches high staining in those cells (HPA: tissue IHC) and CCNK's nuclear location (UniProt O75909). Compare cells within the same section: HPA reports cell-specific levels, so a uniformly dark field is less informative than identifiable stained nuclei (general IHC practice). |
| Strong, convincing cytoplasmic or membrane staining dominates while nuclei remain weak. | Treat this as a localisation mismatch requiring investigation: CCNK is nuclear (UniProt O75909), and HPA describes ubiquitous nuclear tissue staining (HPA: tissue IHC). Review controls and detection conditions before scoring that signal as CCNK; morphology alone cannot identify the cause (general IHC practice). |
| Adipocytes, liver cholangiocytes, or splenic red-pulp cells stain strongly. | These particular cell populations were not detected in the supplied HPA tissue observations (HPA: tissue IHC). Suspect cross-reactivity or endogenous detection activity, and assess a no-primary control (general IHC practice). Do not call the entire adipose, liver, or spleen section negative. |
| Brown signal coats stroma, cytoplasm, or most of the section without crisp nuclear boundaries. | Diffuse background prevents a confident nuclear call (UniProt O75909: nucleus; general IHC practice). Compare a no-primary control and inspect blocking, washes, detection reagent, and counterstain (general IHC practice). HPA tissue levels cannot establish which workflow step caused the background. |
| No nuclear signal appears in adrenal glandular cells or placental trophoblasts. | Those are useful expected-positive cell populations because HPA reports high staining there (HPA: tissue IHC). Check section integrity, the catalog antibody's IHC-P instructions, and detection controls before interpreting absence as biology (general IHC practice). HPA's Supported rating is not a guarantee for every specimen. |
💡Expected CCNK appearanceCall a result positive when identifiable nuclei stain in expected cells, with high signal possible in adrenal glandular cells, cortical glia, or placental trophoblasts (HPA: tissue IHC); dominant membrane staining or indiscriminate brown background is suspect (UniProt O75909: nucleus; general IHC practice).
| Tissue and cell selection | HPA reports high staining in five named cell populations, medium staining in appendix, breast, and bronchus, and low staining in several other populations (HPA: tissue IHC). Select and score the specified cells, since a whole-tissue label can obscure these differences. |
| IHC evidence strength | The tissue profile is Supported, with medium agreement between antibody staining and RNA expression (HPA: reliability). HPA000645 has no supplied IHC status; HPA077073 is Supported for IHC (HPA: antibody validation). Interpret discordant staining with this distinction in view. |
| Isoforms and epitope coverage | Four CCNK isoforms are listed (UniProt O75909: isoforms). Their presence alone does not show which isoforms the catalog antibody recognises; an epitope map would be needed to make that claim. Do not explain a cell-level staining difference by isoform choice without supporting evidence. |
| Processing and nuclear context | UniProt lists a single 1–580 CCNK chain, with no signal peptide, propeptide, or transmembrane segment (UniProt O75909: processing and topology). These annotations support a nuclear interpretation; they provide no basis here for predicting a secreted or surface staining pattern. |
| Antigen retrieval | Use the catalog antibody's IHC-P retrieval instructions when available and judge the result against expected nuclear staining (general IHC practice; UniProt O75909: nucleus). No supplied source establishes CCNK-specific fixation sensitivity or predicts which retrieval condition will recover signal. |
| IF/ICC: where should CCNK appear? | HPA reports enhanced nucleoplasmic localisation and Enhanced ICC validation for HPA000645 and HPA077073 (HPA: subcellular location; HPA: antibody validation). Expect nucleoplasmic fluorescence. These ICC findings support localisation interpretation here; IF/ICC procedure belongs in its separate guide. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected-positive nuclei are blank. | The assay may have failed, or the sampled cells may be unsuitable; HPA reports high staining in adrenal glandular cells and placental trophoblasts (HPA: tissue IHC). | Verify the cell identity and section, run an appropriate positive control, and follow the catalog antibody's IHC-P retrieval and dilution instructions (general IHC practice). Do not infer a CCNK-specific fixation effect from this result. |
| Nuclear signal is faint throughout a control section. | Detection conditions may be weak, although HPA's high categories describe particular cell populations rather than every cell in a tissue (HPA: tissue IHC; general IHC practice). | Confirm that the expected cells are present; review the catalog IHC-P dilution, detection reagents, and development time while retaining a no-primary control (general IHC practice). |
| Cytoplasm or cell borders stain more strongly than nuclei. | The dominant compartment conflicts with CCNK's nuclear annotation and HPA's nuclear tissue profile (UniProt O75909: subcellular location; HPA: tissue IHC). | Check morphology against the counterstain, compare a no-primary control, and reassess antibody specificity before scoring the pattern as CCNK (general IHC practice). |
| Brown staining appears in expected-negative cell populations. | Adipocytes, cholangiocytes, and splenic red-pulp cells are listed as not detected (HPA: tissue IHC); apparent signal may reflect cross-reactivity or endogenous detection activity (general IHC practice). | Identify the stained cell type precisely, examine a no-primary control, and check the relevant endogenous-activity block for the detection chemistry (general IHC practice). |
| Diffuse precipitate obscures nuclear boundaries. | Background or excessive development can prevent compartment scoring (general IHC practice); HPA's nuclear profile does not explain a diffuse deposit (HPA: tissue IHC). | Inspect no-primary and reagent controls, washing, chromogen development, and counterstain; repeat only after the nuclei can be distinguished (general IHC practice). |
| A test tissue disagrees with an expected intensity category. | HPA reports a Supported profile with medium antibody–RNA consistency, and intensity categories apply to specified cells (HPA: reliability; HPA: tissue IHC). | Record the exact cell population and nuclear fraction, compare an expected-positive control in the same run, and describe the discrepancy without assigning an unsupported tissue-wide CCNK status (general IHC practice). |
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 placenta first and expect nuclear staining in trophoblastic cells (HPA: High in placental trophoblastic cells; UniProt O75909: nucleus). Use adipose tissue as a biological comparison, focusing on adipocytes (HPA: Not detected in adipocytes); on the placenta slide, cells without specific nuclear signal can serve as internal background comparators, but no target-negative cell population is established there by the supplied HPA rows.
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) slide, a host-matched nonimmune rabbit IgG control for the catalog rabbit pAb (caption: Rabbit pAb), and a matched CCNK-knockout specimen as a biological negative. Block endogenous peroxidase for chromogenic detection and assess blood-associated background in placenta (standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence, and the selected A06254 paraffin-section caption does not report a fixative (A06254 caption: fixative unreported). The caption uses high-pressure retrieval in 10 mM citrate, pH 6.0, at 1:100 in breast cancer; whether placental staining depends on that retrieval condition requires testing (A06254 caption). The supplied evidence does not establish that frozen sections or IF are easier for this application; in placenta, blood-associated peroxidase can complicate chromogenic interpretation (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
CCNK is predominantly nuclear, so evaluate chromogenic staining by cell type and compartment alongside matched controls (UniProt O75909; HPA tissue IHC).
How should I retrieve CCNK antigen when nuclear staining is weak?
Use heat-mediated retrieval in 10 mM citrate buffer at pH 6.0 before chromogenic IHC (datasheet A06254). The selected paraffin-section image used high-pressure retrieval with this buffer and a 1:100 primary dilution, although its fixative was not reported (caption A06254). If nuclei stain weakly, first confirm that the retrieval vessel reached and maintained its intended temperature, then compare a modestly longer heating interval on adjacent sections (standard IHC practice). Keep section thickness, antibody dilution, detection chemistry and development time constant during that comparison (standard IHC practice). Judge improvement by sharper nuclear signal without increased diffuse background, using the same control tissue across runs (HPA tissue IHC; standard IHC practice).
Could fixation explain weak or uneven CCNK staining?
Target-specific fixation sensitivity for CCNK is unknown from the supplied evidence; the selected paraffin-section caption does not state a fixative (caption A06254). Record the fixative, time before fixation and fixation duration for each specimen before attributing weak staining to CCNK biology (standard IHC practice). Compare similarly processed sections with the same pH 6.0 citrate retrieval and 1:100 primary dilution used in the selected image (caption A06254; standard IHC practice). Poorly preserved nuclear detail or staining confined to well-preserved regions warrants a processing review alongside antibody controls (standard IHC practice). Do not infer a CCNK-specific fixation effect from its nuclear location, phosphorylation sites or tissue staining pattern (UniProt O75909; HPA tissue IHC).
Where should convincing CCNK staining appear in a paraffin section?
Prioritise staining within intact nuclei: CCNK is annotated as nuclear, and tissue IHC shows a ubiquitous nuclear pattern (UniProt O75909; HPA tissue IHC). Its enhanced nucleoplasmic location provides a useful compartment check, although chromogenic sections may not resolve finer nuclear structures (HPA subcellular; standard IHC practice). Compare tumour and surrounding cells against identifiable nuclei in the same section, rather than scoring diffuse cytoplasmic colour as equivalent signal (standard IHC practice). High staining has been reported in adrenal glandular cells and cerebral-cortex glial cells, while breast glandular cells show medium staining (HPA tissue IHC). Use the selected breast-cancer image at 1:100 as an application example, not a universal intensity standard (caption A06254; standard IHC practice).
Can this stain distinguish CCNK isoforms or phosphorylation states?
CCNK has 4 annotated isoforms, but the supplied IHC caption does not identify an antibody epitope or establish isoform selectivity (UniProt O75909; caption A06254). UniProt lists phosphoserines at residues 324, 328, 329 and 340; this does not establish phospho-specific detection by the catalog antibody (UniProt O75909; caption A06254). Treat a positive nucleus as evidence consistent with CCNK staining, not identification of a particular isoform or modification (UniProt O75909; standard IHC interpretation). For a specific molecular claim, obtain the antibody epitope and validation data, then compare staining with an independently validated assay or perturbation (standard IHC practice). Keep retrieval and detection conditions matched when comparing those specimens (standard IHC practice).
How should I check CCNK localisation in a multiplex IF experiment?
For the separate IF/ICC application, pair CCNK with a marker identifying the expected cell type and a nuclear counterstain so nucleoplasmic signal can be assigned to individual cells (HPA subcellular; standard IF practice). HPA reports nucleoplasmic CCNK in IF/ICC images from MCF-7, U-251MG, SiHa and U2OS cells, but those images do not validate this catalog antibody for IF (HPA subcellular; standard IF interpretation). Select a fluorophore channel with low background in the specimen and inspect unstained tissue for autofluorescence before multiplex scoring (standard IF practice). CCNK has no annotated transmembrane segment, so use permeabilisation appropriate for access to an intracellular nuclear epitope, then compare with a matched omitted-primary control (UniProt O75909; standard IF practice).
What should I change if the chromogenic stain obscures nuclei?
First compare a no-primary control with the stained section to separate detection-system colour from antibody-dependent colour (standard IHC practice). If both show widespread brown signal, review endogenous peroxidase blocking and shorten DAB development under otherwise matched conditions (standard IHC practice). If only the antibody section is noisy, optimise blocking and wash steps, then titrate around the caption's 1:100 primary dilution using adjacent sections (caption A06254; standard IHC practice). Inspect whether excess colour follows tissue edges, folds or damaged regions before calling it CCNK expression (standard IHC practice). Preserve the documented pH 6.0 citrate retrieval while changing one background-related variable at a time (datasheet A06254; standard IHC practice).
How can I quantify CCNK staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and nuclear region before scoring, because CCNK is nuclear and HPA tissue staining varies by cell type (UniProt O75909; HPA tissue IHC). Report the percentage of positive intact nuclei and, when intensity is reproducible, a nuclear H-score using intensity grades 0–3 and a 0–300 scale (standard IHC practice). Normalise positive-cell counts to all evaluable nuclei in the specified compartment, or report positive nuclei per mm² of viable tissue (standard IHC practice). Exclude folds, necrosis and edge artefact with the same rules across samples, and hold retrieval, imaging and DAB development constant (standard IHC practice). Include control sections in each staining run to monitor scoring drift (standard IHC practice).
How do I distinguish true CCNK positivity from staining artefact?
A convincing result places signal in intact nuclei within the cell population being scored, consistent with CCNK's nuclear annotation and HPA's ubiquitous nuclear tissue pattern (UniProt O75909; HPA tissue IHC). Strong cytoplasmic colour alone, especially in folds, damaged edges or necrotic areas, warrants review before a positive call (standard IHC practice). Compare the primary-antibody section with a no-primary control; colour shared by both can indicate endogenous enzyme activity or detection background (standard IHC practice). HPA reports no detection in adipocytes, liver cholangiocytes and splenic red-pulp cells, but these observations do not make every such cell a universal negative control (HPA tissue IHC; standard IHC interpretation). Interpret intensity cautiously because HPA rates agreement between staining and RNA expression as medium (HPA tissue IHC).
Boster reagents
Best CCNK / Cyclin-K IHC Antibodies
The catalog antibody has real IHC images from paraffin-embedded human breast cancer and mouse spinal cord (A06254 image captions). IF/ICC image data are unreported (catalog: no IF images).
Real IHC data
Anti-Cyclin-K CCNK Antibody
Cat # A06254
A06254 has IHC images of paraffin-embedded human breast cancer and mouse spinal cord, each stained at 1:100 (A06254 image captions). Its listed applications are IHC and WB, and its listed reactivity is Human, Mouse, and Rat (catalog: A06254 applications and reactivity).
Which to pick: Choose A06254 for tissue IHC: it is a rabbit polyclonal antibody with paraffin-section IHC images in human and mouse tissue (catalog: A06254 host; A06254 image captions). For cross-species work, Human, Mouse, and Rat are listed as reactive, but the supplied IHC images cover Human and Mouse only (catalog: A06254 reactivity; A06254 image captions). No IF/ICC application or image is listed, so there is no supported IF/ICC pick here; the IHC captions specify paraffin embedding and citrate retrieval, while the fixative is unreported (catalog: A06254 applications and IF images; A06254 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 across tissues (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: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A06254); Caveat: Adipocytes may lack detectable staining (HPA tissue IHC); Isoform / epitope: 4 isoforms; epitope effects unknown (UniProt); Regulation: Widely expressed; highest in testis (UniProt). 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.