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CCDC69 / Coiled-coil domain-containing protein 69 · IHC design guide
Design Immunohistochemistry for CCDC69
Plan CCDC69 IHC in paraffin sections with the IHC-validated antibody at 1–5 μg/mL (datasheet: A17261). Expect cytoplasmic staining in most cell types, but interpret intensity cautiously because tissue staining has low agreement with RNA expression (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CCDC69 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: cytoplasm (HPA tissue IHC); mitosis: spindle/midbody (UniProt) | |
| Staining pattern | Cytoplasmic staining in most cell types (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Adipose tissue |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Staining has low agreement with RNA expression (HPA tissue IHC) | |
| Regulation | High in duodenum, pancreas and prostate (UniProt) | |
| Isoform / epitope | 0 annotated isoforms; epitope site unspecified (UniProt) |
Section 1
Recommended CCDC69 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with three published CCDC69 IHC workflows (PMC7922363; PMC11992169; PMC10571395).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A17261); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Chicken anti-CCDC69, 1-5 μg/mL (datasheet A17261) |
| 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 | CCDC69-positive staining in glandular cells of adrenal gland (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in most cell types. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page protocol); a published breast-tissue workflow used citrate at 95 °C for 15 min (PMC10571395).
Section 2
What Is the Expected CCDC69 Staining Pattern?
In paraffin-section IHC, expect predominantly cytoplasmic staining in many cell types, including glandular cells and respiratory epithelium (HPA: tissue IHC). CCDC69 also localizes to the spindle and midbody during cell division (UniProt A6NI79: subcellular location). It has no transmembrane segment (UniProt A6NI79: topology). Interpret any pattern cautiously: HPA rates tissue IHC reliability Uncertain because staining has low consistency with RNA expression (HPA: tissue IHC reliability).
| Cytoplasmic signal in glandular cells or respiratory epithelium. | This fits the broad tissue profile: glandular cells stain at Medium level in adrenal gland, appendix, breast and cervix; bronchial respiratory epithelial cells stain at Medium level (HPA: tissue IHC). It is a plausible positive pattern, but HPA's Uncertain reliability limits how confidently staining alone identifies CCDC69 (HPA: tissue IHC reliability). |
| Predominantly nuclear signal, or a membrane-only pattern, in paraffin sections. | Reassess the result against the reported cytoplasmic tissue-IHC profile and UniProt spindle/midbody localization (HPA: tissue IHC; UniProt A6NI79: subcellular location). A different compartment can indicate nonspecific staining or a preparation-dependent pattern (general IHC practice). HPA reports plasma-membrane localization in ICC-IF, but that assay does not establish a membrane-only tissue-IHC pattern (HPA: subcellular ICC-IF). |
| Strong staining in adipocytes, while the expected cells show little signal. | HPA reports adipocytes as Not detected in adipose tissue (HPA: tissue IHC). Check whether the signal follows the cells or the detection chemistry; off-target antibody binding or endogenous detection activity can produce misleading cell-specific staining (general IHC practice). This tissue comparison is provisional because HPA rates CCDC69 tissue IHC Uncertain (HPA: tissue IHC reliability). |
| Broad haze, precipitate, or similar color across cells and surrounding tissue. | Treat an indiscriminate pattern as background until controls show cell-associated staining (general IHC practice). Review blocking, washes and chromogen development, and inspect a no-primary control for detection-system background (general IHC practice). HPA's cytoplasmic profile concerns cellular staining; it does not validate diffuse background as CCDC69 (HPA: tissue IHC). |
| No signal in glandular cells of a tissue listed as Medium by HPA. | First verify that the sampled section contains the listed cell type and that the detection run worked (HPA: tissue IHC; general IHC practice). Then review antibody dilution, retrieval and detection controls using the validated IHC procedure (general IHC practice). A single negative section cannot settle expression because HPA's tissue-IHC reliability is Uncertain (HPA: tissue IHC reliability). |
💡Expected CCDC69 appearanceCall a result provisionally positive when cell-associated cytoplasmic color is visible in the reported glandular or respiratory epithelial cells at a moderate, interpretable level; isolated nuclear color, adipocyte staining or diffuse haze warrants investigation (HPA: tissue IHC, Medium/Not detected and Uncertain reliability; general IHC practice).
| Tissue-IHC evidence quality | HPA describes cytoplasmic expression in most cell types but assigns Uncertain reliability owing to low agreement between antibody staining and RNA expression (HPA: tissue IHC). Use its Medium and Not detected entries as comparisons, not absolute pass/fail standards. |
| Cell and tissue selection | Listed Medium examples include adrenal, appendix, breast and cervical glandular cells and bronchial respiratory epithelium; adipocytes are Not detected (HPA: tissue IHC). UniProt's high tissue-expression list is a separate observation and does not supply cell-level IHC scores (UniProt A6NI79: tissue specificity). |
| Assay-specific localization | HPA tissue IHC reports cytoplasmic staining, whereas HPA ICC-IF approves plasma-membrane localization with additional Golgi and actin-filament localization (HPA: tissue IHC; HPA: subcellular ICC-IF). Judge paraffin-section chromogenic results against tissue-IHC observations; investigate discordance before assigning an IF compartment to IHC. |
| Topology and mitotic context | CCDC69 has no annotated transmembrane segment (UniProt A6NI79: topology). UniProt describes movement along interpolar microtubules in anaphase and concentration at the midbody in telophase (UniProt A6NI79: subcellular location). Such structures depend on finding cells in the relevant division stage (general IHC practice). |
| Antibody validation scope | HPA043648 is listed as IHC Uncertain, while HPA052896 is listed as ICC Approved with no IHC status supplied (HPA: antibodies). An ICC approval alone cannot validate a paraffin-section result; assess the IHC antibody and its tissue controls in the intended assay (general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected glandular cells are blank. | The section may lack the scored cell population, or the staining run may have failed (HPA: tissue IHC; general IHC practice). | Confirm cell identity and inspect a working positive control; then check retrieval, antibody dilution and detection against the IHC-validated procedure (general IHC practice). |
| Only nuclei or tissue edges are intensely colored. | The distribution departs from HPA's cytoplasmic tissue profile; edge effects or nonspecific signal are possible (HPA: tissue IHC; general IHC practice). | Compare interior cells with edges and a no-primary control; review section handling, washes and chromogen development (general IHC practice). |
| Adipocytes appear strongly positive. | HPA lists adipocytes as Not detected; endogenous detection activity or off-target binding may explain apparent color (HPA: tissue IHC; general IHC practice). | Check a no-primary control and cell morphology, then compare staining in the listed Medium cell populations within a controlled run (general IHC practice; HPA: tissue IHC). |
| Color obscures cell boundaries across the section. | Excess detection product or insufficient washing can create diffuse background (general IHC practice). | Inspect the no-primary control, shorten chromogen development if excessive, and review blocking and wash steps (general IHC practice). |
| Two tissue sections give different apparent intensities. | The sampled cell types may differ, and HPA's tissue-IHC profile has Uncertain reliability (HPA: tissue IHC). | Score comparable cells with the same detection conditions and controls; report compartment, cell type and intensity separately (general IHC practice). |
| Does ICC-IF membrane staining establish a positive IHC result? | HPA approves a mainly plasma-membrane ICC-IF location, while its tissue-IHC profile is cytoplasmic and Uncertain (HPA: subcellular ICC-IF; HPA: tissue IHC). | Evaluate the paraffin section with tissue-IHC controls and its own compartment readout; consult the separate IF/ICC guide for fluorescence assay design (general IHC/IF practice). |
Experiment evidence
IHC & IF images showing CCDC69 staining
A17261 · IHCImmunohistochemistry of CCDC69 in human lung tissue with CCDC69 antibody at 5 μg/mL.
A17261 · IFImmunofluorescence of CCDC69 in human lung tissue with CCDC69 antibody at 20 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000198624-CCDC69/tissue/cerebral+cortex#imid_11871663
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000198624-CCDC69/tissue/colon#imid_11871581
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000198624-CCDC69/tissue/liver#imid_11871590
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000198624-CCDC69/tissue/kidney#imid_11871599
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA052896; green = CCDC69, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA052896; green = CCDC69, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CCDC69 IHC & IF
🧪Run breast first: glandular cells should stain (HPA: Medium in breast glandular cells). Use adipose tissue as the negative tissue (HPA: Not detected in adipocytes); adipocytes within the breast section should remain at background, providing a same-slide comparison (HPA: Not detected in adipocytes).
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 ICC-IF images show CCDC69 in A-431, U-251MG, U2OS, with annotated localisation: Plasma membrane (approved) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, an isotype control matched to the primary antibody’s host species, immunoglobulin class and clonality, and CCDC69 knockout tissue if available (standard IHC practice). Quench endogenous peroxidase for chromogenic detection and, if using biotin-based detection, block endogenous biotin (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence, and the A17261 lung IHC caption does not state a fixative (caption: fixative unreported). Antigen-retrieval dependence is unreported; compare retrieved and unretrieved paraffin sections during optimization (standard IHC practice). Frozen sections are not established as easier; IF/ICC can help assess localization (HPA: ICC-IF images available), while adipocytes in breast sections provide a check for tissue background (HPA: Not detected in adipocytes).
HPA tissue IHC evidence for CCDC69
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Low consistency between antibody staining and RNA expression data. Pending external verification.). 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 | Glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Caudate | Glial cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CCDC69 IHC Tips
Troubleshoot CCDC69 staining by checking retrieval, compartment patterns and controls before comparing chromogenic IHC scores (UniProt A6NI79; HPA: tissue IHC reliability uncertain).
How should I retrieve CCDC69 in paraffin sections when staining is weak?
Start with citrate buffer at pH 6.0 for heat-induced antigen retrieval at 95–98 °C for 20 minutes (page retrieval setting). Allow sections to cool in buffer, then compare staining with a section processed without retrieval to identify heat-related background or tissue damage (standard IHC practice). The selected antibody has a human lung tissue-IHC image at 5 µg/mL, but its caption does not report retrieval conditions or fixative (caption A17261). Judge any improvement by cellular pattern and control staining, because independent tissue-IHC staining has low consistency with RNA data and remains uncertain (HPA: tissue IHC reliability uncertain).
Could fixation explain weak or uneven CCDC69 staining?
Target-specific fixation sensitivity is unknown: the selected tissue-IHC caption gives no fixative, and no fixation comparison is supplied (caption A17261). For paraffin-section IHC, record fixation conditions and compare sections processed consistently before changing antibody concentration or retrieval, since fixation can alter epitope accessibility (standard IHC practice). Check whether weak staining follows section edges, folds or poorly preserved regions, and exclude those regions from scoring (standard IHC practice). Do not attribute a fixation effect to CCDC69’s spindle association or phosphoserines at residues 154 and 241; those annotations do not establish fixation sensitivity (UniProt A6NI79).
Which cellular staining pattern should I accept as plausible for CCDC69?
In tissue IHC, assess cytoplasmic staining first, because HPA reports cytoplasmic expression in most cell types (HPA: tissue IHC profile). A concentrated midzone or midbody signal in dividing cells is biologically plausible: CCDC69 localizes along interpolar microtubules in early anaphase and at the midbody during telophase (UniProt A6NI79). HPA cell imaging also reports plasma membrane, Golgi and actin-filament localization, so compartment calls need orthogonal confirmation rather than automatic rejection (HPA: subcellular). Compare candidate patterns with morphology and suitable control sections; the tissue-IHC evidence is rated uncertain because staining and RNA expression have low consistency (HPA: tissue IHC reliability uncertain).
How can I troubleshoot an epitope-dependent CCDC69 staining discrepancy?
Check the antibody’s stated immunogen or epitope before comparing staining across reagents; no epitope sequence is supplied here for the selected antibody (caption A17261). The supplied protein record lists a 296-amino-acid precursor, a chain spanning residues 2–296, no annotated isoforms and no transmembrane segment (UniProt A6NI79). Phosphoserines at residues 154 and 241 are annotated, but their effect on this antibody’s binding is unknown (UniProt A6NI79). If two antibodies disagree, compare their documented epitope regions and tissue patterns under matched processing, then seek an independent specificity control before assigning the discrepancy to an isoform or modification (standard IHC practice).
How should I check CCDC69 localisation by multiplex IF after tissue IHC?
Use multiplex IF as a separate localisation check, pairing CCDC69 with a marker of the expected cell type, such as an epithelial marker when examining bronchial respiratory epithelium (HPA: bronchus respiratory epithelial cells, medium staining). Choose spectrally separated fluorophores and favor a far-red channel when tissue autofluorescence obscures shorter wavelengths (standard IF practice). Optimize permeabilisation against the antibody’s documented epitope: access to a cytoplasmic epitope generally needs permeabilisation, whereas an exposed cell-surface epitope may not (standard IF practice; UniProt A6NI79: no transmembrane segment). HPA cell imaging reports membrane, Golgi and actin-filament patterns; compare those patterns with compartment markers and controls before transferring an IF interpretation to chromogenic IHC (HPA: subcellular).
What should I change if chromogenic CCDC69 staining is diffuse?
Run a section without primary antibody to identify detection-reagent background, and include a peroxidase block before chromogenic development (standard IHC practice). Titrate the primary around the selected image’s 5 µg/mL concentration while holding retrieval, incubation and DAB development constant; that image documents staining in human lung tissue, not an optimized range (caption A17261). Check blocking, wash stringency and DAB development time if diffuse color persists, then inspect whether the signal follows tissue edges or damaged areas (standard IHC practice). Interpret widespread cytoplasmic color cautiously: HPA reports cytoplasmic expression in most cell types but rates tissue-IHC reliability uncertain (HPA: tissue IHC profile and reliability).
How should I score CCDC69 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring; for example, score bronchial respiratory epithelial cells separately from neighboring cells when using that tissue (HPA: bronchus respiratory epithelial cells, medium staining). Record the percentage of positive cells and staining intensity, then calculate an H-score if intensity categories are reproducible (standard IHC practice). Normalize counts to the number of evaluable cells, or report positive-cell density per mm² of viable tissue, using the same region-selection rules across sections (standard IHC practice). Exclude folds, edges and necrotic areas, and report controls and uncertainty because HPA tissue staining has low consistency with RNA expression (standard IHC practice; HPA: tissue IHC reliability uncertain).
When is a CCDC69-positive IHC result convincing rather than artefactual?
A credible result has reproducible cellular staining in intact tissue, passes the no-primary control and follows a plausible compartment pattern (standard IHC practice). Cytoplasmic expression is reported in most cell types, while spindle-midzone and midbody localization is expected in dividing cells (HPA: tissue IHC profile; UniProt A6NI79). Scrutinize isolated nuclear-only color, section-edge enhancement, necrotic regions and color retained without primary antibody; the latter can indicate endogenous enzyme activity or detection background (standard IHC practice). Avoid declaring a cell type definitively positive from one image: the selected caption shows human lung tissue at 5 µg/mL, and broader HPA tissue-IHC evidence remains uncertain (caption A17261; HPA: tissue IHC reliability uncertain).
Boster reagents
Best CCDC69 / Coiled-coil domain-containing protein 69 IHC Antibodies
A17261 has IHC and IF images from human lung tissue (catalog image captions). Human, mouse and rat reactivity is listed, but IHC and IF validation is reported only for human samples (datasheet).
Real IHC data
Anti-CCDC69 Antibody
Cat # A17261
A17261 has an IHC image of human lung tissue at 5 μg/mL (IHC image caption). A17261 also has an IF image of human lung tissue at 20 μg/mL (IF image caption).
Which to pick: For tissue IHC, choose A17261: it is listed for IHC-P, with a human lung IHC image at 5 μg/mL; the image caption does not report the fixative (catalog applications; IHC image caption). For IF, choose A17261 based on its human lung IF image; ICC is not listed as an application (IF image caption; catalog applications). For mouse or rat work, A17261 lists both species as reactive, but its datasheet reports IHC and IF validation only in human samples (catalog reactivity; datasheet).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry A6NI79 (CCD69_HUMAN, Coiled-coil domain-containing protein 69).
- Human Protein Atlas. CCDC69 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. CCDC69 subcellular location (ICC-IF): Mainly localized to the plasma membrane. In addition localized to the Golgi apparatus and actin filaments..
- Human Protein Atlas. CCDC69 antibody validation summary (2 antibodies).
- Study on CCDC69 interfering with the prognosis of patients with breast cancer through PPAR signal pathway. European journal of histochemistry : EJH 2021 — PMC7922363.
- Identification of M1 macrophage infiltration-related genes for immunotherapy in Her2-positive breast cancer based on bioinformatics analysis and machine learning. Scientific reports 2025 — PMC11992169.
- High expression of CCDC69 is correlated with immunotherapy response and protective effects on breast cancer. BMC cancer 2023 — PMC10571395.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15372022 — UniProt-cited evidence.
- PubMed PMID:20962590 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CCDC69 (Coiled-coil domain-containing protein 69). Expected localisation Tissue: cytoplasm (HPA tissue IHC); mitosis: spindle/midbody (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A17261 at 1-5 μg/mL (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: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet A17261); 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-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 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: Chicken anti-CCDC69, 1-5 μg/mL (datasheet A17261); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Staining has low agreement with RNA expression (HPA tissue IHC); Isoform / epitope: 0 annotated isoforms; epitope site unspecified (UniProt); Regulation: High in duodenum, pancreas and prostate (UniProt). Evidence: UniProt A6NI79, HPA tissue IHC · Uncertain, HPA ICC-IF · Mainly localized to the plasma membrane. In addition localized to…, Datasheet A17261, PMC7922363, PMC11992169. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CCDC69/immunohistochemistry-ihc-guide.