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DDX6 / Probable ATP-dependent RNA helicase DDX6 · IHC design guide
Design Immunohistochemistry for DDX6
Plan DDX6 staining in paraffin sections using the observed cytoplasmic tissue pattern (HPA tissue IHC). Compare glandular cells with strong reported staining and account for possible stress related relocalization (HPA tissue IHC; UniProt).
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 | Predominantly cytoplasmic in tissue sections (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining 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 conditions consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Cellular stress may shift DDX6 to granules (UniProt) | |
| Regulation | Abundant in most tissues (UniProt) | |
| Isoform / epitope | 0 annotated isoforms; no signal peptide or propeptide (UniProt) |
Section 1
Recommended DDX6 IHC & IF Protocols
The catalog antibody’s IHC-P protocol uses EDTA pH 8.0 retrieval (datasheet: A03826-1). One published DDX6 protocol for paraffin sections is shown below (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); consider citrate pH 6.0 when adapting the published testicular tissue protocol (PMC6028756).
Section 2
What Is the Expected DDX6 Staining Pattern?
DDX6 should show predominantly cytoplasmic staining in IHC, including glandular cells in appendix, colon, duodenum and rectum, and squamous epithelial cells in esophagus (HPA: general cytoplasmic expression; High in those cells). It is an intracellular protein with no transmembrane segment (UniProt P26196 topology). HPA rates tissue IHC as Approved, with medium consistency between staining and RNA data and external verification pending (HPA: reliability).
| Cytoplasmic staining in appendix or colon glandular cells, with relatively little signal in nearby unstained areas. | This fits the reported IHC pattern: general cytoplasmic expression and High staining in those glandular cells (HPA: tissue IHC). Compare cell compartments within the section; the reported High level describes HPA observations, not a required absolute intensity in every run. |
| Nuclear staining dominates while cytoplasmic staining is absent or weak. | Recheck specificity and scoring before calling this the expected IHC result (HPA: general cytoplasmic expression). Nuclear DDX6 is possible through import with EIF4ENIF1/4E-T, so a nuclear signal alone does not prove artefact (UniProt P26196: subcellular location). |
| An unexpected cell population stains strongly while an expected positive population does not. | Consider cross-reactivity or endogenous detection activity (general IHC practice). HPA reports High staining in appendix glandular cells and esophageal squamous epithelial cells, but also reports abundant expression across most tissues; an unlisted cell type is not automatically negative (HPA: tissue IHC; UniProt P26196: tissue specificity). |
| Color spreads across tissue compartments or appears on areas lacking identifiable cells. | This is diffuse background rather than a convincing intracellular pattern (general IHC practice; HPA: general cytoplasmic expression). Review blocking, washing and chromogen development using appropriate controls (general IHC practice). |
| No cytoplasmic signal appears in a known-positive tissue section. | Treat this as a failed or inconclusive run until controls pass (general IHC practice). Appendix, colon and duodenum glandular cells are reported High by HPA, although HPA's tissue IHC assessment has only medium staining–RNA consistency and awaits external verification (HPA: tissue IHC; reliability). |
💡Expected DDX6 appearanceCall positive a predominantly cytoplasmic signal in the reported High-staining cells, such as colon glandular cells; dominant nuclear color or diffuse color without cell boundaries calls for investigation (HPA: tissue IHC; UniProt P26196: nuclear localization; general IHC practice).
| Cell population and tissue | HPA reports High staining in several glandular epithelia, esophageal squamous epithelial cells and ovarian follicle cells, but Low staining in glial cells, myocytes, smooth muscle cells and chondrocytes (HPA: tissue IHC). Select comparators by cell type as well as organ. |
| Intracellular location | DDX6 lacks a transmembrane segment; UniProt places it in cytoplasm, P-bodies and sometimes nucleus (UniProt P26196: topology; subcellular location). Judge the main IHC pattern against HPA's general cytoplasmic profile (HPA: tissue IHC). |
| Physiological redistribution | DDX6 can relocalize to stress granules under cellular stress (UniProt P26196: subcellular location). The supplied tissue IHC observations do not establish that stress explains any particular paraffin-section pattern (HPA: tissue IHC). |
| Antibody evidence | Three listed antibodies have Approved IHC status; one also has Supported ICC status (HPA: antibodies HPA024201, HPA026644, CAB004668). HPA's tissue-level assessment still reports medium staining–RNA consistency and pending external verification (HPA: reliability). |
| IF/ICC: what pattern is expected? | Cytoplasmic bodies are the supported main location, with supported cytosol staining; plasma membrane and midbody locations are uncertain (HPA: subcellular ICC-IF). This IF/ICC observation does not establish a required granule appearance in chromogenic tissue IHC (HPA: subcellular ICC-IF; tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| A positive-control section has no detectable staining. | The run may have failed at primary-antibody incubation, detection or chromogen development (general IHC practice). HPA reports High glandular-cell staining in appendix and colon (HPA: tissue IHC). | Check reagent preparation, incubation and detection controls, then repeat with a reported High-staining tissue (general IHC practice; HPA: tissue IHC). Do not infer DDX6 absence from this run. |
| The negative control develops color. | Endogenous activity, nonspecific secondary binding or excess chromogen development may contribute (general IHC practice). | Check the no-primary control and the detection system; adjust blocking and development according to the assay controls (general IHC practice). Reassess DDX6 staining only after background is controlled. |
| Nuclei are the strongest feature throughout a section. | This differs from HPA's general cytoplasmic IHC profile, although UniProt records possible nuclear import (HPA: tissue IHC; UniProt P26196: subcellular location). | Compare cytoplasm and nuclei in a reported High-staining cell population and verify controls before assigning a specific nuclear pattern (HPA: tissue IHC; general IHC practice). |
| Staining is broadly diffuse and cell boundaries are hard to distinguish. | Nonspecific background or overdevelopment can obscure compartment scoring (general IHC practice). | Review no-primary control, blocking, washes and chromogen timing; score localization only when individual cells remain interpretable (general IHC practice). |
| A low-staining comparator appears as strong as a reported positive. | The chosen populations may differ from HPA observations, or background may compress the contrast (HPA: tissue IHC; general IHC practice). | Compare matched cell populations and controls: HPA reports Low staining in skeletal-muscle myocytes and High staining in colon glandular cells (HPA: tissue IHC). Investigate persistent discordance before interpreting it biologically. |
| A punctate IF/ICC result seems inconsistent with broad cytoplasmic IHC. | The assays report different levels of spatial detail: HPA describes cytoplasmic bodies in ICC-IF and general cytoplasmic expression in tissue IHC (HPA: subcellular ICC-IF; tissue IHC). | Interpret each preparation against its own HPA reference pattern; do not require resolvable puncta in chromogenic paraffin sections (HPA: subcellular ICC-IF; tissue IHC). |
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 show strong DDX6 staining (HPA: Colon glandular cells, High). HPA detects DDX6 in all 45 scored tissues, so no negative tissue or internal negative cell population is established; no-primary and isotype slides provide the negative comparison, with only background signal expected in any unstained cells (HPA: no negative rows; UniProt P26196: abundant expression in most tissues).
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 rabbit IgG isotype control (selected-SKU caption: rabbit primary antibody). Where available, use DDX6 knockout material as a biological negative, and block endogenous peroxidase before HRP/DAB detection (selected-SKU caption: HRP/DAB detection).
⚠️Feasibility: The selected-SKU paraffin-section example used heat retrieval in EDTA at pH 8.0, but its fixative is unreported; no target-specific fixation window or fixation effect is supplied (selected-SKU tissue-IHC caption). That example supports testing the reported retrieval condition, but does not establish that retrieval is required or that frozen sections or IF are easier (selected-SKU tissue-IHC caption). Score cytoplasmic signal with attention to punctate bodies, and check diffuse glandular background against the negative controls (UniProt P26196: cytoplasm/P-body; HPA: cytoplasmic bodies supported; HPA: Colon glandular cells, High).
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, and cell-specific controls (datasheet A03826-1; HPA tissue IHC; UniProt P26196).
How should I optimise retrieval when DDX6 staining is weak?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A03826-1). The selected image used this retrieval before incubation with 2 μg/ml catalog antibody overnight at 4°C; its caption does not report the fixative (datasheet A03826-1). If staining remains weak, vary heating time in a small section series while keeping antibody concentration, detection, and development conditions constant (standard IHC practice). Check a positive tissue region alongside each condition, and reject settings that increase diffuse background or damage section morphology (HPA: high glandular-cell staining in colon; standard IHC practice).
Could fixation explain weak or patchy DDX6 staining?
Target-specific fixation sensitivity is unknown because the selected paraffin-section caption does not state a fixative (datasheet A03826-1). Record the actual fixative, fixation duration, and processing history for each specimen before comparing staining intensity across cases (standard IHC practice). Compare sections processed alike, then assess whether changes in retrieval improve cytoplasmic staining while preserving morphology and limiting background (HPA: general cytoplasmic expression; standard IHC practice). Do not infer fixation tolerance from DDX6 topology, phosphorylation, or the HPA tissue pattern; those records describe protein features and observed staining, not controlled fixation comparisons (UniProt P26196; HPA tissue IHC).
Is nuclear or diffuse staining compatible with DDX6?
Expect predominantly cytoplasmic staining in tissue sections, while recognising that DDX6 is associated with P-bodies and other cytoplasmic ribonucleoprotein granules (HPA: general cytoplasmic expression; UniProt P26196). Nuclear localisation is biologically possible through interaction with EIF4ENIF1/4E-T, but a nuclear-only chromogenic pattern needs independent confirmation (UniProt P26196; standard IHC practice). Compare signal with the corresponding cell morphology and with a no-primary control before assigning puncta or nuclear colour to DDX6 (standard IHC practice). Treat uniform staining of unrelated structures or section edges as possible detection artefact until the pattern persists across well-preserved regions (standard IHC practice).
How can epitope uncertainty affect DDX6 IHC interpretation?
The record lists a 483-amino-acid DDX6 chain, no isoforms, no signal peptide, and no transmembrane segment (UniProt P26196). It places the ATP-binding helicase region at residues 127–298, the C-terminal helicase region at 308–468, and a reported phosphothreonine at residue 36 (UniProt P26196). Without a mapped antibody epitope, these annotations cannot establish whether retrieval or phosphorylation changes antibody binding (UniProt P26196; standard IHC practice). If a result conflicts with expected cytoplasmic staining, compare an independently validated epitope reagent and matched control sections before attributing the difference to a DDX6 variant or modification (HPA: general cytoplasmic expression; standard IHC practice).
How should I assess DDX6 localisation by multiplex IF?
For the separate IF/ICC workflow, pair DDX6 with a marker that identifies the expected cell type, such as a glandular-cell marker when assessing colon sections (HPA: high in colon glandular cells; standard IF practice). Choose spectrally separated fluorophores and a detection channel that avoids prominent tissue autofluorescence, checking each channel with single-stain controls (standard IF practice). DDX6 lacks a transmembrane segment and is mainly associated with cytoplasmic bodies and cytosol, so use permeabilisation sufficient to access intracellular epitopes while checking that punctate structures remain discernible (UniProt P26196; HPA subcellular; standard IF practice). The epitope and IF fixation requirements for this catalog antibody are unspecified here; establish them with appropriate IF controls (datasheet A03826-1; standard IF practice).
What causes brown signal outside expected DDX6-positive cells?
First compare the stained section with a no-primary control and inspect whether brown colour follows tissue folds, edges, damaged areas, or endogenous pigment (standard IHC practice). The selected paraffin-section image used 10% goat serum blocking, a peroxidase-conjugated secondary, and DAB development (datasheet A03826-1). A peroxidase block and controlled DAB development are general chromogenic IHC steps; neither establishes DDX6 specificity by itself (standard IHC practice). If background persists, titrate the catalog antibody around the documented 2 μg/ml condition and compare cytoplasmic cell staining against background under identical development conditions (datasheet A03826-1; HPA: general cytoplasmic expression; standard IHC practice).
How should I score DDX6 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and cytoplasmic compartment before scoring, because tissue IHC shows general cytoplasmic expression with cell-dependent intensity (HPA tissue IHC). For a chromogenic intensity endpoint, report the percentage of positive cells and an H-score using intensity grades 0–3; record the scoring thresholds and apply them uniformly (standard IHC practice). Normalise counts to the number of evaluable cells in the same cell class, or report positive-cell density per mm² of viable annotated tissue (standard IHC practice). Exclude folds, necrosis, and section edges consistently, and compare only sections with comparable processing and DAB development (standard IHC practice).
How can I distinguish genuine DDX6 signal from staining artefact?
Look for reproducible cytoplasmic signal in morphologically intact cells, consistent with the HPA tissue profile and DDX6 association with cytoplasmic P-bodies (HPA tissue IHC; UniProt P26196). High staining in appendix or colon glandular cells provides a useful reference pattern, while low staining in skeletal-muscle myocytes cautions against treating every brown structure as positive (HPA tissue IHC). Investigate nuclear-only signal, staining confined to section edges or necrosis, and colour present in a no-primary control before assigning DDX6 positivity (standard IHC practice). Endogenous peroxidase or pigment can mimic DAB signal, so interpret cellular localisation together with negative controls and preserved morphology (standard IHC practice).
Boster reagents
Best DDX6 / Probable ATP-dependent RNA helicase DDX6 IHC Antibodies
A03826-1 has human paraffin-section IHC data (IHC caption); A03826-1 and M03826-1 have human ICC/IF images (IF captions). Both list Human, Mouse, Rat reactivity (catalog).
Real IHC data
Anti-DDX6 Antibody ®
Cat # A03826-1
Real IF data
Anti-DDX6 Antibody ® (monoclonal, 8G6)
Cat # M03826-1
A03826-1 will render with an IHC image from a human stomach cancer paraffin section; its application list also includes IF and ICC (A03826-1 IHC caption; catalog). M03826-1 will render with an IF image from MCF-7 cells and is listed for IF and ICC (M03826-1 IF caption; catalog).
Which to pick: Choose A03826-1 for chromogenic IHC on human paraffin sections: its caption documents EDTA retrieval at pH 8.0, 2 μg/ml primary antibody, and DAB detection; the fixative is unreported (A03826-1 IHC caption). Choose M03826-1 for ICC/IF: it is mouse monoclonal clone 8G6, and its IF caption documents MCF-7 cells; IHC is absent from its application list (catalog; M03826-1 IF caption). For cross-species planning, both SKUs list Human, Mouse, Rat reactivity, while their rendered images show human samples (catalog; 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 Predominantly cytoplasmic 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 conditions consistent across sections (standard IHC practice; not target-specific); Caveat: Cellular stress may shift DDX6 to granules (UniProt); Isoform / epitope: 0 annotated isoforms; no signal peptide or propeptide (UniProt); Regulation: Abundant in most tissues (UniProt). 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.