This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
DDX6 / Probable ATP-dependent RNA helicase DDX6 · IHC design guide
Design Immunohistochemistry for DDX6
Plan paraffin IHC for DDX6 using cytoplasmic staining as the tissue benchmark (HPA tissue IHC). Colon glandular cells offer a high-staining reference (HPA tissue IHC); the IHC-validated antibody has a stated range of 2–5 µg/mL (datasheet A03826-1).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
DDX6 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic staining in tissue sections (HPA tissue IHC) | |
| Staining pattern | General cytoplasmic staining; high in glandular cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A03826-1) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Cellular stress can shift DDX6 to stress granules (UniProt) | |
| Regulation | Broad expression; tissue intensity varies (HPA tissue IHC) | |
| Isoform / epitope | No annotated isoforms or cleavage products (UniProt) |
Section 1
Recommended DDX6 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is paired with a published chromogenic DDX6 protocol for human testicular tissue (PMC6028756).
| Sample | Paraffin-embedded human stomach cancer tissue; fixative not specified (datasheet A03826-1) |
| Fixation | Image fixative and duration unreported (datasheet A03826-1); 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 A03826-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A03826-1) |
| Primary antibody | Rabbit anti-DDX6, 2-5μg/ml (datasheet A03826-1) |
| Primary incubation | Overnight at 4 °C (datasheet A03826-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A03826-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | DDX6-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet: A03826-1); the published testicular protocol used citrate pH 6.0 (PMC6028756).
Section 2
What Is the Expected DDX6 Staining Pattern?
DDX6 should show general cytoplasmic staining in paraffin sections, with strong staining expected in several glandular and squamous epithelial populations (HPA: tissue IHC). It is a nonmembrane protein that also occupies P-bodies and can enter the nucleus (UniProt P26196: topology and localization). Treat tissue patterns as a guide: HPA rates its IHC reliability Approved, with medium consistency against RNA data and external verification pending (HPA: reliability).
| Cytoplasmic staining is strongest in appendix or colon glandular cells; hematoxylin leaves nuclei distinct. | This fits the reported general cytoplasmic pattern and High staining in those cells (HPA: tissue IHC). Compare staining within the same section before scoring intensity, since chromogenic signal also reflects the IHC workflow (general IHC practice). |
| Staining is predominantly nuclear, with little cytoplasmic signal in an otherwise positive tissue. | Investigate the compartment pattern: cytoplasm is the principal tissue IHC finding (HPA: tissue IHC). Nuclear entry is documented, so nuclear signal alone is not proof of an artefact (UniProt P26196: localization); check controls and staining distribution (general IHC practice). |
| Strong staining appears in glial cells or myocytes while expected epithelial cells are weak. | HPA reports Low staining in hippocampal and caudate glia and skeletal myocytes, but High staining in several glandular epithelia (HPA: tissue IHC). Check for cross-reactivity or endogenous chromogenic activity before assigning the unexpected signal to DDX6 (general IHC practice). |
| Brown deposit covers stroma, lumina, or much of the section without a clear cell pattern. | Diffuse deposit does not match the reported cellular cytoplasmic pattern (HPA: tissue IHC). Review blocking, washes, detection reagents and the negative control for nonspecific staining or endogenous activity (general IHC practice). |
| No signal appears in appendix or colon glandular cells. | Those cells are reported High and are useful positive comparators (HPA: tissue IHC). A blank result calls for review of the positive control, antigen retrieval, primary antibody dilution and detection steps; it cannot establish absent DDX6 on its own (general IHC practice). |
💡Expected DDX6 appearanceCall a positive result when glandular or squamous epithelial cells show strong cytoplasmic staining (HPA: High in listed epithelia; general cytoplasmic expression); diffuse extracellular deposit or exclusive membrane outlines warrant investigation (general IHC practice; HPA: plasma membrane localization uncertain in ICC-IF).
| Choice of tissue comparator | Appendix, colon, duodenum, endometrium, gallbladder and rectum glandular cells are reported High; skeletal myocytes and selected glia are Low (HPA: tissue IHC). Use the named cell population when judging a control, since whole-section staining can hide that distinction (general IHC practice). |
| Granules and compartment resolution | DDX6 occupies cytoplasmic P-bodies and can move to stress granules under cellular stress (UniProt P26196: localization). Tissue IHC is reported as generally cytoplasmic (HPA: tissue IHC); do not require individually resolved puncta to accept a chromogenic section (general IHC practice). |
| Antibody evidence | HPA024201, HPA026644 and CAB004668 have Approved IHC status (HPA: antibody validation). That status supports their reported tissue patterns, while HPA's medium RNA consistency and pending external verification limit how firmly an unexpected pattern can be assigned to DDX6 (HPA: reliability). |
| Topology and processing | DDX6 has no transmembrane segment, signal peptide or propeptide (UniProt P26196: topology and processing). A dominant membrane-outline pattern deserves checking against cytoplasmic positive cells; the uncertain plasma membrane observation in ICC-IF does not establish that pattern for tissue IHC (HPA: subcellular). |
| IF/ICC: what should a positive cell show? | Expect cytoplasmic bodies with additional cytosol signal (HPA: ICC-IF, supported). Plasma membrane and midbody assignments are uncertain (HPA: ICC-IF). This describes cellular localization; it does not define a staining protocol or override the tissue IHC pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive glandular cells are blank. | The expected High signal is missing (HPA: appendix and colon glandular cells); the failed step is undetermined. | Confirm tissue and cell identity, then review retrieval, primary antibody dilution, detection reagents and a positive control in the same run (general IHC practice). |
| Cytoplasm is faint across all tissues. | A weak run may obscure the general cytoplasmic pattern (HPA: tissue IHC); HPA supplies no DDX6-specific fixation-sensitivity finding (HPA: supplied tissue IHC record). | Compare known High cells and run controls; review section handling, retrieval and detection as general IHC variables without attributing the result to a DDX6-specific fixation effect (general IHC practice). |
| Only nuclei stain strongly. | The balance differs from general cytoplasmic tissue staining (HPA: tissue IHC), although nuclear import is possible (UniProt P26196: localization). | Check whether cytoplasmic positive cells stain, inspect the negative control, and repeat with the IHC-validated antibody if the compartment pattern remains discordant (general IHC practice; HPA: IHC Approved). |
| Brown signal is widespread outside cells. | Nonspecific deposit or endogenous chromogenic activity can obscure a cellular pattern (general IHC practice); HPA reports general cytoplasmic expression (HPA: tissue IHC). | Inspect a negative control, blocking and wash steps, then score only interpretable cellular staining (general IHC practice). |
| Low-reference cells appear stronger than expected positive cells. | Cell identity, section variability or an off-target reaction may explain the mismatch (general IHC practice); HPA lists glia and myocytes as Low and several glandular populations as High (HPA: tissue IHC). | Verify the named cell populations on the counterstain and compare controls. Treat a persistent reversal as uncertain, given HPA's medium RNA consistency and pending external verification (HPA: reliability). |
Experiment evidence
IHC & IF images showing DDX6 staining
A03826-1 · IHCIHC analysis of DDX6 using anti-DDX6 antibody (A03826-1). DDX6 was detected in a paraffin-embedded section of human stomach cancer tissue. H
A03826-1 · IFIF analysis of DDX6 using anti-DDX6 antibody (A03826-1). DDX6 was detected in immunocytochemical section of U2OS cells. Enzyme antigen retri
M03826-1 · IFIF analysis of DDX6 using anti-DDX6 antibody (M03826-1). DDX6 was detected in immunocytochemical section of MCF-7 cells. Enzyme antigen retr
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000110367-DDX6/tissue/cerebral+cortex#imid_6525448
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000110367-DDX6/tissue/colon#imid_6524870
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000110367-DDX6/tissue/liver#imid_6524855
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000110367-DDX6/tissue/kidney#imid_6524856
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA024201; green = DDX6, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA024201; green = DDX6, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for DDX6 IHC & IF
🧪Run colon first: its glandular cells should stain (HPA: High in colon glandular cells). HPA lists no negative tissue and detects DDX6 in all 45 scored tissues (HPA: negative rows empty); use no-primary and isotype controls, and expect any unstained cells on the positive slide to show counterstain only, without DAB signal, without assigning a cell type as DDX6-negative.
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: None in HPA: DDX6 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show DDX6 in A-431, U-251MG, U2OS, ASC52telo, Sperm, KOLF2.1J, with annotated localisation: Cytoplasmic bodies (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide and a concentration-matched nonimmune rabbit IgG isotype control (selected-SKU caption: rabbit primary antibody); a DDX6 knockout specimen processed alongside them would provide a biological negative. Quench endogenous peroxidase before HRP/DAB detection, especially when evaluating blood or inflammatory cells in colon (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU caption: fixative not stated). Heat retrieval in EDTA at pH 8.0 was used for that IHC example, but whether DDX6 staining depends on it is unreported (selected-SKU caption: EDTA retrieval); colon mucus may retain background stain (standard IHC practice). The supplied evidence does not establish whether frozen sections or IF are easier; ICC-IF images support cytoplasmic bodies as a localization check (HPA: cytoplasmic bodies supported).
HPA tissue IHC evidence for DDX6
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium 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 |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Endometrium | Glandular cells | High | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: DDX6 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced DDX6 IHC Tips
Troubleshoot DDX6 staining in paraffin sections by checking retrieval, cytoplasmic localisation, controls and scoring before interpreting chromogenic signal.
Which retrieval conditions should I try first for DDX6 in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 (datasheet A03826-1). The selected image used this condition on a paraffin section of human stomach cancer, followed by 2 μg/ml primary antibody overnight at 4°C (datasheet A03826-1). If staining is weak, adjust heating time or cooling within a controlled comparison while keeping antibody concentration and detection conditions constant (standard IHC practice). Compare signal in glandular cells with tissue morphology and a no-primary control; strong glandular staining is documented in several tissues, but that pattern does not establish retrieval performance in every specimen (HPA: tissue IHC).
How should I troubleshoot fixation-related loss of DDX6 staining?
The selected DDX6 image identifies a paraffin section but does not report its fixative (datasheet A03826-1). Target-specific sensitivity to fixation is therefore unknown; do not infer it from DDX6 topology, phosphorylation or tissue staining patterns (UniProt P26196; HPA: tissue IHC). For a weak batch, compare sections with documented fixation and processing histories while holding pH 8.0 EDTA retrieval, 2 μg/ml antibody and detection settings constant (datasheet A03826-1; standard IHC practice). Check morphology and a known staining control alongside the test section, because poor preservation or inconsistent processing can complicate interpretation of a negative chromogenic result (standard IHC practice).
Where should convincing DDX6 staining appear in tissue sections?
Expect predominantly cytoplasmic staining in chromogenic tissue IHC; the tissue profile reports general cytoplasmic expression (HPA: tissue IHC). DDX6 also associates with cytoplasmic P-bodies and can occur in the nucleus, so a nuclear component is biologically possible but needs corroboration in the section (UniProt P26196: subcellular location). Cytoplasmic bodies are supported by cell imaging, whereas plasma membrane and midbody assignments are uncertain; those imaging observations do not define a chromogenic tissue pattern (HPA: subcellular). Compare suspicious signal with cell boundaries, counterstain and no-primary control, and review glandular cells where high tissue staining has been reported (HPA: tissue IHC; standard IHC practice).
Could an isoform or epitope issue explain discordant DDX6 staining?
The supplied record lists a single 483-aa DDX6 chain and 0 isoforms, so it provides no alternative isoform to explain a discordant tissue pattern (UniProt P26196: processing and isoforms). It places the ATP-binding helicase region at residues 127–298, the C-terminal helicase region at 308–468, and a phosphothreonine at residue 36 (UniProt P26196: domains and modified residues). The antibody epitope is not supplied, so these features cannot establish whether retrieval or phosphorylation alters its binding (datasheet A03826-1; UniProt P26196). If results disagree, compare antibody specificity controls and matched processing before assigning an epitope-based explanation (standard IHC practice).
How can IF help assess a puzzling chromogenic DDX6 pattern?
Use IF as a separate localisation check when chromogenic IHC leaves cytoplasmic signal hard to assign to a cell type (standard IF practice). Multiplex DDX6 with a cytokeratin marker to identify epithelial glandular cells, which show high DDX6 staining in several tissues (HPA: tissue IHC; standard IF practice). Choose a far-red fluorophore where tissue autofluorescence interferes with shorter wavelengths, and inspect single-label controls before interpreting overlap (standard IF practice). DDX6 has no transmembrane segment and is associated with cytoplasmic P-bodies, so permeabilisation should permit access to a cytoplasmic epitope; the antibody's exact epitope is unreported (UniProt P26196: topology and subcellular location; datasheet A03826-1).
What should I check when DDX6 DAB staining is diffuse?
First separate cytoplasmic signal from diffuse deposit by comparing stained sections with a no-primary control and checking whether DAB follows intact cells (HPA: tissue IHC; standard IHC practice). The selected image used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and a peroxidase-based DAB detection system (datasheet A03826-1). Check peroxidase blocking, washing and chromogen development as general sources of background; these steps do not establish DDX6 specificity (standard IHC practice). If background persists, titrate primary antibody and detection exposure together with a positive control, while keeping retrieval at pH 8.0 EDTA initially (datasheet A03826-1; standard IHC practice).
How should I score heterogeneous DDX6 staining across a section? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue compartment and cell population before scoring, because DDX6 has a general cytoplasmic tissue profile with high staining in selected glandular populations (HPA: tissue IHC). For chromogenic sections, record the percentage of positive cells and staining intensity, then calculate an H-score from the same predefined cell population; use positive-cell density per mm² when cell abundance itself is relevant (standard IHC practice). Normalise to viable nucleated cells or measured tissue area, and exclude folds, edges and necrotic regions using consistent criteria (standard IHC practice). Keep retrieval, imaging and DAB development comparable across batches, and report cytoplasmic and nuclear scores separately if both are present (datasheet A03826-1; UniProt P26196: subcellular location; standard IHC practice).
When is an apparent DDX6-positive area likely to be artefactual?
A convincing result follows intact cell morphology and shows a predominantly cytoplasmic pattern consistent with tissue IHC observations (HPA: tissue IHC; standard IHC practice). Strong staining confined to section edges, folds or necrosis warrants comparison with adjacent intact tissue and a no-primary control (standard IHC practice). Diffuse extracellular DAB or signal reproduced without primary antibody raises concern for detection artefact, including endogenous enzyme activity (standard IHC practice). Nuclear signal alone deserves cautious interpretation because DDX6 can enter the nucleus, while its established tissue profile is generally cytoplasmic; confirm the compartment and cell identity before calling it DDX6-specific (UniProt P26196: subcellular location; HPA: tissue IHC).
Boster reagents
Best DDX6 / Probable ATP-dependent RNA helicase DDX6 IHC Antibodies
The catalog shows DDX6 IHC in human paraffin stomach cancer tissue and IF/ICC in human U2OS and MCF-7 cells (catalog image captions). Both antibodies list human, mouse and rat reactivity (catalog reactivity).
Real IHC data
Anti-DDX6 Antibody ®
Cat # A03826-1
Real IF data
Anti-DDX6 Antibody ® (monoclonal, 8G6)
Cat # M03826-1
A03826-1 has IHC data from human paraffin stomach cancer tissue and IF/ICC data from U2OS cells (A03826-1 image captions). M03826-1 has IF/ICC data from MCF-7 cells and no listed IHC application (M03826-1 image caption; catalog applications).
Which to pick: Choose A03826-1 for paraffin-section IHC: its own caption shows staining of human stomach cancer tissue at 2 μg/ml after heat retrieval in EDTA, pH 8.0; the fixative is unreported (A03826-1 IHC caption). For IF/ICC, M03826-1 is a mouse monoclonal with an MCF-7 image, while A03826-1 also has a U2OS IF/ICC image (catalog host and clone; M03826-1 and A03826-1 IF captions). Both list human, mouse and rat reactivity, but the supplied IHC and IF images show human samples only; A03826-1 is the pictured tissue-IHC choice for work involving those species (catalog reactivity; image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P26196 (DDX6_HUMAN, Probable ATP-dependent RNA helicase DDX6).
- Human Protein Atlas. DDX6 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. DDX6 subcellular location (ICC-IF): Mainly localized to the cytoplasmic bodies. In addition localized to the plasma membrane, cytosol and midbody..
- Human Protein Atlas. DDX6 antibody validation summary (3 antibodies).
- Imsnc761 and DDX6 synergistically suppress cell proliferation and promote apoptosis via p53 in testicular embryonal carcinoma cells. Bioscience reports 2018 — PMC6028756.
- DEAD-Box Helicase 6 Blockade in Brain-Derived Aβ Oligomers From Alzheimer's Disease Patients Attenuates Neurotoxicity. MedComm 2025 — PMC12018770.
- DEAD-Box Protein RNA-Helicase DDX6 Regulates the Expression of HER2 and FGFR2 at the Post-Transcriptional Step in Gastric Cancer Cells. International journal of molecular sciences 2018 — PMC6073682.
- PubMed PMID:1579499 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:1394235 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for DDX6 (Probable ATP-dependent RNA helicase DDX6). Expected localisation Cytoplasmic staining in tissue sections (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A03826-1). Validated IHC antibody: A03826-1 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human stomach cancer tissue; fixative not specified (datasheet A03826-1); 2. Fixation: Image fixative and duration unreported (datasheet A03826-1); 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 A03826-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A03826-1); 8. Primary antibody: Rabbit anti-DDX6, 2-5μg/ml (datasheet A03826-1); 9. Primary incubation: Overnight at 4 °C (datasheet A03826-1). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Cellular stress can shift DDX6 to stress granules (UniProt); Isoform / epitope: No annotated isoforms or cleavage products (UniProt); Regulation: Broad expression; tissue intensity varies (HPA tissue IHC). Evidence: UniProt P26196, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the cytoplasmic bodies. In addition localized to…, Datasheet A03826-1, PMC6028756. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/DDX6/immunohistochemistry-ihc-guide.