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DDIT4 / DNA damage-inducible transcript 4 protein · IHC design guide
Design Immunohistochemistry for DDIT4
Plan chromogenic IHC-P for DDIT4 with the catalog antibody, starting at 5 μg/mL as shown for mouse tissue (datasheet: A02019). Assess cytoplasmic staining in glandular cells and use adipocytes, where staining was undetected, as a tissue comparison (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
DDIT4 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 | General cytoplasmic tissue staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in glandular cells (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 | RNA levels may not predict tissue staining (HPA tissue IHC) | |
| Regulation | Responds to hypoxia and DNA damage (UniProt) | |
| Isoform / epitope | No annotated isoforms; one 1–232 chain (UniProt) |
Section 1
Recommended DDIT4 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with published DDIT4 staining protocols for lung adenocarcinoma and gastric cancer sections (PMC8489140; PMC11929655).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A02019); 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 | Rabbit anti-DDIT4, 5 μg/mL (datasheet A02019) |
| 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 | DDIT4-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page antigen retrieval). The gastric study used a 10 min autoclave retrieval (PMC11929655).
Section 2
What Is the Expected DDIT4 Staining Pattern?
DDIT4 should show predominantly cytoplasmic staining, consistent with supported cytosolic localisation in ICC-IF and a cytosolic or mitochondrial assignment in UniProt (HPA: general cytoplasmic IHC expression; HPA: cytosol supported; UniProt Q9NX09: subcellular location). Expect strong signal in the cell populations HPA scores High, including adrenal glandular cells and bone marrow hematopoietic cells (HPA: tissue IHC). HPA rates tissue IHC Approved, with medium consistency between staining and RNA expression (HPA: tissue IHC reliability).
| Cytoplasmic chromogen is clear in adrenal glandular cells or bone marrow hematopoietic cells, with visible cellular boundaries (HPA: High in these cells). | This fits the reported tissue and compartment pattern (HPA: general cytoplasmic expression). Compare signal within the named cell population, since a tissue-wide average can obscure differences between neighbouring cells (general IHC interpretation). |
| Signal is confined to nuclei, with little or no cytoplasmic staining (HPA: general cytoplasmic expression; HPA: cytosol supported). | A nuclear-only pattern conflicts with the reported localisation (HPA: cytosol supported; UniProt Q9NX09: cytosol and mitochondrion). Check counterstain, chromogen deposits and control slides before calling it DDIT4 (general IHC practice). |
| Adipocytes stain strongly while the expected positive cells remain unstained (HPA: adipocytes Not detected; HPA: selected cells High). | Treat this as a suspect cell-type pattern, not proof of DDIT4 expression (HPA: tissue IHC). Cross-reactivity or endogenous detection activity are possible explanations to investigate with antibody and detection controls (general IHC practice). |
| Colour spreads across extracellular space or coats many unrelated structures without distinct cellular staining (HPA: general cytoplasmic expression). | This is diffuse background rather than the reported cytoplasmic pattern (HPA: tissue IHC). Review reagent-only controls, blocking, washes and detection timing to locate nonspecific signal (general IHC practice). |
| No signal appears in a section containing identifiable adrenal glandular cells or bone marrow hematopoietic cells (HPA: High in these cells). | A negative result in a reported high-staining population calls for a run check (HPA: tissue IHC; general IHC practice). It does not alone establish absent DDIT4: compare a known-positive section and inspect staining controls before interpreting the specimen (general IHC practice). |
💡Expected DDIT4 appearanceCall a positive result when discrete cytoplasmic staining is evident in an HPA High cell population, such as adrenal glandular cells; nuclear-only colour or strong adipocyte staining warrants investigation (HPA: general cytoplasmic expression; HPA: adrenal glandular cells High; HPA: adipocytes Not detected).
| Which compartment should guide scoring? (HPA: general cytoplasmic expression) | Score cellular cytoplasm first (HPA: tissue IHC; HPA: cytosol supported). UniProt also lists mitochondrion, so its annotation does not make every punctum diagnostic; compare the full pattern with controls (UniProt Q9NX09: subcellular location; general IHC practice). |
| Which cells offer useful positive and low-signal comparisons? (HPA: tissue IHC) | Adrenal glandular cells and bone marrow hematopoietic cells are High; adipocytes are Not detected (HPA: tissue IHC). Liver cholangiocytes are listed as low, so a faint result there is less informative than a failed high-staining control (HPA: tissue IHC; general IHC interpretation). |
| How much weight should the tissue atlas carry? (HPA: IHC Approved) | The tissue result is Approved but has medium consistency with RNA expression (HPA: tissue IHC reliability). Use its named cell patterns as expectations, then judge the actual section and controls; the atlas rating is not a guarantee for every specimen (general IHC practice). |
| Does DDIT4 topology predict a surface or shed staining pattern? (UniProt Q9NX09: topology) | No transmembrane segment, signal peptide or propeptide is annotated; the chain spans residues 1–232 (UniProt Q9NX09: topology and processing). These annotations support an intracellular expectation, without identifying an antibody epitope or predicting retrieval performance (UniProt Q9NX09; general IHC interpretation). |
| IF/ICC: where should signal appear? (HPA: subcellular ICC-IF) | HPA supports cytosolic localisation and lists A-431, U2OS and hTCEpi among imaged cell lines (HPA: subcellular ICC-IF). That observation informs compartment interpretation; IF/ICC preparation and controls belong in the separate IF/ICC guide (HPA: subcellular ICC-IF). |
| Situation | Likely cause | Next action |
|---|---|---|
| A reported high-staining population is blank (HPA: adrenal glandular cells High). | The run may have failed, or the specimen may differ from the atlas example (general IHC practice; HPA: tissue IHC). | Confirm the named cells are present, run a known-positive section and check primary antibody and detection controls before scoring absence (general IHC practice). |
| Only nuclei appear positive (HPA: general cytoplasmic expression). | Counterstain overlap, precipitate or nonspecific detection can imitate cellular signal (general IHC practice). | Inspect a no-primary control and the chromogen pattern; require convincing cytoplasmic signal for a DDIT4 call (general IHC practice; HPA: cytosol supported). |
| Adipocytes show prominent colour (HPA: adipocytes Not detected). | The pattern conflicts with HPA and may reflect cross-reactivity or endogenous detection activity (HPA: tissue IHC; general IHC practice). | Compare a no-primary control and an HPA High cell population on the same run; investigate the detection system before assigning adipocyte positivity (general IHC practice; HPA: tissue IHC). |
| Background obscures cell boundaries (HPA: general cytoplasmic expression). | Nonspecific reagent binding, insufficient washing or excess chromogen development can reduce contrast (general IHC practice). | Review blocking, washes and development time using appropriate controls; reassess whether cytoplasmic staining remains distinct (general IHC practice; HPA: tissue IHC pattern). |
| Signal is weak in liver cholangiocytes (HPA: low in cholangiocytes). | A low-staining population offers limited sensitivity as a positive run control (HPA: tissue IHC; general IHC interpretation). | Check a reported High population, such as adrenal glandular cells, before changing the staining conditions or calling the run negative (HPA: tissue IHC; general IHC practice). |
| A proposed retrieval change is attributed to DDIT4 fixation sensitivity (HPA: tissue IHC; UniProt Q9NX09). | The supplied sources report staining and protein annotations, without target-specific fixation or retrieval effects (HPA: tissue IHC; UniProt Q9NX09). | Treat retrieval adjustment as a general IHC optimisation; compare controlled sections and report the observed result without claiming a DDIT4-specific fixation effect (general IHC practice). |
Experiment evidence
IHC & IF images showing DDIT4 staining
A02019 · IHCImmunohistochemistry of RTP801 in mouse kidney tissue with RTP801 antibody at 5 μg/mL.
A02019 · IFImmunofluorescence of RTP801 in mouse kidney tissue with RTP801 antibody at 20 μg/mL. Red: RTP801 Antibody (A02019) Blue: DAPI staining
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000168209-DDIT4/tissue/cerebral+cortex#imid_8491556
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000168209-DDIT4/tissue/colon#imid_8490842
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000168209-DDIT4/tissue/liver#imid_8490827
HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000168209-DDIT4/tissue/pancreas#imid_8490778
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA034508; green = DDIT4, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA034508; green = DDIT4, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for DDIT4 IHC & IF
🧪Run adrenal gland first and score its glandular cells, which should stain (HPA: High in adrenal gland glandular cells). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes); on the adrenal slide, assess any unstained non-glandular cells as internal background, without assuming they are biologically negative (standard IHC practice).
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 DDIT4 in A-431, U2OS, hTCEpi, with annotated localisation: Cytosol (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; a concentration-matched isotype control of the primary antibody’s host species and clonality; and, if available, a DDIT4 knockout specimen or validated peptide block (standard IHC practice). Block endogenous peroxidase and inspect adrenal sections for pigment that could be mistaken for chromogenic signal (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and antigen-retrieval dependency are unreported in the supplied evidence; the selected A02019 mouse-kidney IHC caption reports 5 µg/mL but does not report a fixative (selected A02019 caption). Frozen sections and IF cannot be judged easier from these data, though cytosolic signal has been observed by ICC-IF (HPA: Cytosol, supported). On adrenal sections, distinguish pigment from the chromogenic signal when scoring (standard IHC practice).
HPA tissue IHC evidence for DDIT4
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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 → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Cervix | Glandular cells | High | 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 DDIT4 IHC Tips
Troubleshoot chromogenic DDIT4 IHC in paraffin sections using compartment, cell type and control patterns to evaluate staining.
How should I retrieve DDIT4 when paraffin sections stain weakly?
Start with citrate buffer at pH 6.0 for heat induced retrieval at 95–98 °C for 20 minutes (page retrieval setting). Cool sections consistently before blocking, then compare a small retrieval time series on adjacent sections while keeping antibody concentration and detection conditions fixed. The catalog image shows mouse kidney staining at 5 µg/mL, but its caption does not report retrieval conditions (A02019 IHC caption). Score cytoplasmic signal against tissue background because DDIT4 is reported in the cytosol and mitochondrion (UniProt Q9NX09 subcellular location). If staining remains weak, test an alternative retrieval buffer as a fallback, alongside the citrate condition and a no-primary control.
Could fixation be masking DDIT4 in my paraffin sections?
DDIT4-specific sensitivity to fixation is unknown from the supplied evidence; the selected tissue image does not state its fixative (A02019 IHC caption). Record fixative, fixation duration and tissue thickness for each specimen, then compare matched sections processed with the same retrieval and detection conditions. If material permits, vary fixation duration in a controlled pilot and assess both cytoplasmic staining and tissue morphology before changing antibody concentration. Avoid treating a stronger signal alone as proof of better preservation: the expected pattern is broadly cytoplasmic (HPA tissue IHC profile), with cytosolic and mitochondrial locations reported (UniProt Q9NX09 subcellular location). Include a no-primary control in each processing batch.
What staining pattern should count as plausible DDIT4 localisation?
Prioritise cytoplasmic staining: tissue IHC reports general cytoplasmic expression, and cell imaging supports a cytosolic location (HPA tissue IHC profile; HPA subcellular). A mitochondrial component is also annotated, so fine cytoplasmic puncta may merit follow-up with an independently validated mitochondrial marker (UniProt Q9NX09 subcellular location). DDIT4 has no annotated transmembrane segment, making an exclusively crisp plasma membrane outline difficult to reconcile with the record (UniProt Q9NX09 topology). Compare staining within intact cells and across adjacent sections before assigning puncta to an organelle. Strong nuclear-only staining should trigger review of retrieval, counterstain, antibody specificity and the no-primary control rather than automatic scoring as positive.
Could epitope choice explain inconsistent DDIT4 staining?
The supplied record lists one 232-amino-acid chain, no annotated isoforms and no transmembrane segment (UniProt Q9NX09 processing, isoforms and topology). It also lists phosphorylation at residues 19, 23, 25 and 121, but the catalog image does not identify the antibody epitope (UniProt Q9NX09 modified residues; A02019 IHC caption). Therefore, do not assign a staining difference to a specific phosphosite or isoform without epitope mapping and appropriate validation. Compare adjacent sections under identical retrieval and detection conditions, and document the antibody identifier before comparing studies. If a second antibody is available, require concordant cell and compartment patterns before interpreting discordance as biology.
How can I check DDIT4 localisation by multiplex IF?
Use a separately validated glandular cell marker when examining glandular regions, where high DDIT4 staining is reported in several tissues (HPA tissue IHC positive-cell entries). Choose a spectrally separated far-red channel for DDIT4 when tissue autofluorescence affects shorter wavelengths, and inspect single-stain controls before interpreting overlap. Because DDIT4 is supported in the cytosol and has no annotated transmembrane segment, optimise gentle permeabilisation for intracellular access rather than assuming a surface epitope (HPA subcellular; UniProt Q9NX09 topology). Check whether the chosen antibody is validated for IF and whether its epitope is disclosed; the supplied catalog image documents tissue IHC only (A02019 IHC caption). Keep the no-primary and single-stain controls alongside the multiplex specimen.
What should I adjust when DDIT4 chromogenic staining is diffuse?
First inspect a no-primary section for detection-system colour and a hematoxylin-only section for counterstain that could obscure weak cytoplasmic signal. In a peroxidase and DAB workflow, check the peroxidase block, wash stringency and DAB development time as general IHC controls. Then titrate the antibody around the documented 5 µg/mL mouse kidney image condition, recognising that the caption gives no fixative or retrieval details (A02019 IHC caption). Compare intact cell staining with acellular deposits and section edges under the same exposure and scoring rules. DDIT4 is reported as generally cytoplasmic, so uniform colour over nuclei, stroma and empty spaces warrants troubleshooting (HPA tissue IHC profile).
How should I quantify heterogeneous DDIT4 staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, since DDIT4 tissue staining is generally cytoplasmic (HPA tissue IHC profile). For chromogenic sections, record the percentage of positive cells and intensity categories, then calculate an H-score from 0–300 using the same thresholds across samples. Alternatively, report positive cells per mm² within a predefined region when cell density varies markedly. Normalise counts to viable nucleated cells or annotated tissue area, and analyse comparable cell types separately; high staining is reported in several glandular populations and bone marrow hematopoietic cells (HPA tissue IHC positive-cell entries). Exclude folds, necrosis and edge artefacts before blinded scoring.
How do I distinguish genuine DDIT4 signal from artefact?
Look for reproducible intracellular cytoplasmic signal in intact cells, consistent with the tissue IHC profile and supported cytosolic location (HPA tissue IHC profile; HPA subcellular). Compare cell identity: high staining is reported in several glandular cell populations, while adipocytes are listed as not detected in adipose tissue (HPA tissue IHC positive and negative-cell entries). Treat nuclear-only, sharply membrane-only or extracellular deposits cautiously because the supplied localisation and topology do not support those patterns (UniProt Q9NX09 subcellular location and topology). Edge-restricted colour, necrotic regions and staining in a no-primary section suggest processing or endogenous enzyme artefact. Confirm an unexpected pattern with matched controls and, if available, an independently validated antibody.
Boster reagents
Best DDIT4 / DNA damage-inducible transcript 4 protein IHC Antibodies
A02019 is an anti-DDIT4 antibody with mouse kidney IHC and IF images (A02019 image captions); its listed reactivity covers Human, Mouse, and Rat (A02019 catalog reactivity).
Real IHC data
Anti-RTP801 DDIT4 Antibody
Cat # A02019
A02019 shows mouse kidney IHC staining at 5 μg/mL (A02019 IHC image caption). It also shows mouse kidney IF staining at 20 μg/mL (A02019 IF image caption).
Which to pick: For paraffin-section tissue IHC, choose A02019: it lists IHC-P and shows mouse kidney staining at 5 μg/mL (A02019 application list; A02019 IHC image caption); the caption does not report the fixative (A02019 IHC image caption). For IF, A02019 shows mouse kidney staining at 20 μg/mL; ICC validation is unreported (A02019 IF image caption; A02019 application list). For cross-species work, A02019 lists Human, Mouse, and Rat reactivity and a Rabbit host, while clone information is unreported; its pictured IHC and IF data are from mouse kidney (A02019 catalog reactivity/host/clone; A02019 image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9NX09 (DDIT4_HUMAN, DNA damage-inducible transcript 4 protein).
- Human Protein Atlas. DDIT4 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. DDIT4 subcellular location (ICC-IF): Localized to the cytosol..
- Human Protein Atlas. DDIT4 antibody validation summary (2 antibodies).
- DDIT4 overexpression associates with poor prognosis in lung adenocarcinoma. Journal of Cancer 2021 — PMC8489140.
- DDIT4 promotes gastric cancer proliferation and tumorigenesis through the p53 and MAPK pathways. Cancer communications (London, England) 2018 — PMC6034313.
- Pan-cancer analysis of DDIT4 identifying its prognostic value and function in acute myeloid leukemia. Journal of cancer research and clinical oncology 2024 — PMC10954950.
- Lower cytoplasmic expression of DDIT4 is associated with poor prognosis in gastric cancer patients. Discover oncology 2025 — PMC11929655.
- PubMed PMID:12453409 — UniProt-cited evidence.
- PubMed PMID:11884613 — UniProt-cited evidence.
- PubMed PMID:11230166 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for DDIT4 (DNA damage-inducible transcript 4 protein). Expected localisation General cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A02019 at 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 A02019); 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: Rabbit anti-DDIT4, 5 μg/mL (datasheet A02019); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: RNA levels may not predict tissue staining (HPA tissue IHC); Isoform / epitope: No annotated isoforms; one 1–232 chain (UniProt); Regulation: Responds to hypoxia and DNA damage (UniProt). Evidence: UniProt Q9NX09, HPA tissue IHC · Approved, HPA ICC-IF · cytosol., Datasheet A02019, PMC8489140, PMC11929655. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/DDIT4/immunohistochemistry-ihc-guide.