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DDB1 / DNA damage-binding protein 1 · IHC design guide
Design Immunohistochemistry for DDB1
Plan DDB1 paraffin-section IHC around its broadly cytoplasmic tissue staining (HPA tissue IHC). Compare cytoplasmic and nuclear staining with consistent fixation and matched controls; UV irradiation can recruit DDB1 to nuclear damage sites (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
DDB1 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: cytoplasmic; molecular: cytoplasm and nucleus (HPA tissue IHC; UniProt) | |
| Staining pattern | Cytoplasmic staining across many tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet PB9578) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Smooth muscle |
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 | IHC staining has low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | UV drives nuclear accumulation (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope differences unreported (UniProt) |
Section 1
Recommended DDB1 IHC & IF Protocols
Start with the catalog antibody’s IHC-P protocol (datasheet: PB9578), then compare the published DDB1 IHC conditions below (PMC6947845; PMC9731993; PMC3294142).
| Sample | Paraffin-embedded Mouse Liver tissue; fixative not specified (datasheet PB9578) |
| Fixation | Image fixative and duration unreported (datasheet PB9578); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet PB9578) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9578) |
| Primary antibody | Rabbit anti-DDB1, 0.5-1μg/ml (datasheet PB9578) |
| Primary incubation | Overnight at 4 °C (datasheet PB9578) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9578) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | DDB1-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteTry heat-mediated citrate retrieval at pH 6 first (datasheet: PB9578); the mouse liver study used a different retrieval solution at 121°C for 5 min (PMC3294142).
Section 2
What Is the Expected DDB1 Staining Pattern?
DDB1 is primarily cytoplasmic and can move into nuclei after UV damage (UniProt Q16531). In tissue IHC, expect broad cytoplasmic staining, including glandular, neuronal and squamous epithelial cells (HPA: ubiquitous cytoplasmic profile; listed High cells). HPA rates its tissue IHC evidence Approved but reports low consistency between staining and RNA expression (HPA: reliability description). DDB1 has no transmembrane segment (UniProt Q16531 topology).
| Cytoplasmic staining in colon glandular cells, cerebral cortex neurons or esophageal squamous cells (HPA: High in each). | This fits the reported tissue pattern (HPA: ubiquitous cytoplasmic expression). Judge staining in the named cells and its compartment, rather than requiring every cell on the section to have the same intensity (HPA: cell-level staining entries). |
| Staining is confined to cell borders or extracellular areas, with little cytoplasmic signal. | That distribution conflicts with the tissue IHC profile (HPA: ubiquitous cytoplasmic expression; UniProt Q16531: no transmembrane segment). Recheck morphology and detection controls before calling it DDB1. Nuclear signal alone is less decisive: UniProt also lists the nucleus (UniProt Q16531 subcellular location). |
| Strong staining occurs mainly in smooth muscle cells while listed High cells are faint (HPA: tissue entries). | Investigate cross-reactivity or endogenous chromogen activity (general IHC practice). Smooth muscle cells were Not detected in the supplied HPA profile, but one negative entry is a comparison point, not proof that every smooth muscle specimen must be blank (HPA: smooth muscle). |
| Color covers empty spaces or obscures cell boundaries across the section. | Treat this as background until controls clarify its source (general IHC practice). Widespread color is insufficient evidence for DDB1 even though HPA describes broad expression; the reported pattern is cellular and cytoplasmic (HPA: tissue IHC profile). |
| No signal appears in a known High cell population on the same run (HPA: listed High cells). | A negative result is uninterpretable until staining performance is checked (general IHC practice). Confirm tissue identity and the expected cells, then examine the antibody, detection reagents and a run control. HPA's Approved rating has a low staining-to-RNA consistency caveat (HPA: reliability description). |
💡Expected DDB1 appearanceCall a positive result when named High cell populations show clear cytoplasmic staining above background (HPA: tissue IHC profile); isolated border, extracellular or cell-free color is suspect (UniProt Q16531 topology; general IHC practice).
| Compartment and biological context (UniProt Q16531; HPA: tissue IHC and subcellular profiles) | UniProt reports primarily cytoplasmic DDB1 with nuclear movement after UV irradiation; HPA tissue IHC reports ubiquitous cytoplasmic staining. A nuclear component needs context and controls, rather than automatic rejection (UniProt Q16531; HPA: tissue IHC profile). |
| Tissue and cell choice (HPA: tissue IHC entries) | Use a listed High population, such as colon glandular cells or cerebral cortex neurons, to assess a run. Thyroid and epididymal glandular cells are Low; smooth muscle cells are Not detected in the supplied profile (HPA: tissue IHC entries). |
| Antibody evidence (HPA: antibody validation; tissue IHC reliability) | HPA045174 and CAB032821 are IHC Approved; HPA068456 has no supplied IHC status. The tissue profile is Approved with low staining-to-RNA consistency, so neither status nor a single stain settles specificity (HPA: antibody validation; reliability description). |
| Protein architecture (UniProt Q16531 topology and isoforms) | No transmembrane segment or signal peptide is annotated, and two isoforms are listed (UniProt Q16531). These facts support checking cellular localisation; they do not identify the antibody epitope or predict which isoform it detects (UniProt Q16531; epitope data not supplied). |
| IF/ICC: where should DDB1 appear? (HPA: subcellular profile) | HPA reports enhanced nucleoplasmic localisation in ICC-IF, with additional sperm-piece locations, while tissue IHC is described as cytoplasmic (HPA: subcellular and tissue IHC profiles). Interpret each application against its own evidence; ICC validation does not establish IHC performance (HPA: antibody validation). |
| Situation | Likely cause | Next action |
|---|---|---|
| A listed High population is blank (HPA: tissue IHC entries). | The run may have failed, or the expected cell population may be absent from the section (general IHC practice). | Verify morphology and tissue identity; compare a known-positive section in the same run, then review antibody and detection steps (general IHC practice). |
| Signal appears only at cell borders or outside cells. | The location conflicts with cytoplasmic tissue staining and a protein without a transmembrane segment (HPA: tissue IHC profile; UniProt Q16531 topology). | Check the section against a detection-only control and reassess where the color lies relative to cells (general IHC practice). |
| Smooth muscle is strong, but listed High cells are weak (HPA: tissue IHC entries). | Cross-reactivity or endogenous detection activity is possible (general IHC practice); HPA lists smooth muscle cells as Not detected. | Check a primary-omission control for detection-related color and compare another IHC-validated antibody if available (general IHC practice; HPA: IHC Approved antibodies). |
| Diffuse color makes compartments impossible to score. | Background may arise from nonspecific binding or detection chemistry (general IHC practice). | Review blocking, washing and chromogen development with appropriate controls; score DDB1 only where cellular boundaries and cytoplasm remain discernible (general IHC practice; HPA: cytoplasmic profile). |
| A tissue section shows prominent nuclear staining. | Nuclear DDB1 is biologically plausible, but HPA's tissue IHC summary emphasizes cytoplasm (UniProt Q16531 subcellular location; HPA: tissue IHC profile). | Document the compartment and sample context; compare positive and detection controls before deciding whether the nuclear signal is specific (general IHC practice). |
| An ICC-IF result disagrees with chromogenic tissue IHC. | HPA reports enhanced nucleoplasm in ICC-IF and ubiquitous cytoplasm in tissue IHC; the supplied antibody validation statuses also differ by application (HPA: subcellular, tissue IHC and antibody profiles). | Score each assay against its application-specific reference and antibody validation; do not transfer an ICC-IF localisation call directly to a tissue section (HPA: profiles; general interpretation practice). |
Experiment evidence
IHC & IF images showing DDB1 staining
PB9578 · IHCIHC analysis of DDB1 using anti-DDB1 antibody (PB9578). DDB1 was detected in paraffin-embedded section of Mouse Liver Tissue. Heat mediated
PB9578 · IHCIHC analysis of DDB1 using anti-DDB1 antibody (PB9578). DDB1 was detected in paraffin-embedded section of Rat Liver Tissue. Heat mediated an
PB9578 · IFIF analysis of DDB1 using anti-DDB1 antibody (PB9578). DDB1 was detected in immunocytochemical section of A431 cells. Enzyme antigen retriev
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000167986-DDB1/tissue/cerebral+cortex#imid_7558104
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000167986-DDB1/tissue/colon#imid_7558038
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000167986-DDB1/tissue/liver#imid_7558039
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000167986-DDB1/tissue/kidney#imid_7558040
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA045174; green = DDB1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA045174; green = DDB1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for DDB1 IHC & IF
🧪Run cerebellum first and look for DDB1 staining in Purkinje cells (HPA: High in Purkinje cells). Use smooth muscle cells as the negative tissue control (HPA: Not detected in smooth muscle cells); on the cerebellum slide, cells without specific staining should show only background signal, but the supplied HPA row does not identify a validated internal negative cell type (HPA: cerebellum row).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Smooth muscle (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show DDB1 in A-431, MCF-7, U2OS, A-549, HEK293, Sperm, NIH 3T3, with annotated localisation: Nucleoplasm (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only section, a rabbit IgG isotype control matched to the primary antibody’s clonality, and a matched DDB1 knockout section if available (caption: rabbit anti-DDB1; standard IHC control practice). For chromogenic detection, quench endogenous peroxidase and check for endogenous biotin before using the caption’s biotin-based detection system (caption: biotinylated secondary and SABC with DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected PB9578 paraffin-section caption does not state the fixative (PB9578 tissue-IHC caption). That caption uses citrate retrieval at pH 6 for 20 minutes in mouse liver; it provides a starting condition, but does not establish that retrieval is required in cerebellum (PB9578 tissue-IHC caption). The supplied evidence does not establish that frozen sections or IF are easier than paraffin IHC; HPA reports enhanced nucleoplasmic ICC-IF signal, while endogenous pigment in cerebellar cells should be distinguished from DAB staining by the negative controls (HPA: subcellular ICC-IF; standard IHC practice).
HPA tissue IHC evidence for DDB1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low 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 → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced DDB1 IHC Tips
Use the DDB1 tissue section evidence to set up chromogenic IHC, then evaluate compartment patterns, controls, and staining quality before scoring.
How should I retrieve DDB1 antigen in paraffin sections?
Use heat-mediated antigen retrieval in citrate buffer at pH 6 for 20 minutes (datasheet PB9578). The catalog antibody’s mouse liver section was processed this way, so it is the starting condition for this IHC assay (datasheet PB9578). If staining is weak, compare retrieval duration on serial sections while keeping the antibody and detection steps constant (standard IHC practice). Check whether stronger retrieval improves cellular staining without increasing tissue damage or diffuse background (standard IHC practice). Record heating, cooling, and staining conditions for each section because differences in processing can confound comparisons (standard IHC practice).
How can I assess whether fixation is affecting DDB1 staining?
Target-specific fixation sensitivity is unknown because the selected paraffin-section caption does not state a fixative (datasheet PB9578). Do not treat that image or the reported localisation as evidence that DDB1 tolerates any particular fixation condition (datasheet PB9578; UniProt Q16531 localisation). For IHC troubleshooting, compare sections with documented processing histories using the same citrate pH 6 retrieval and detection workflow (datasheet PB9578; standard IHC practice). Examine nuclear detail, tissue integrity, and background alongside DDB1 signal before changing retrieval conditions (standard IHC practice). Report fixation as unrecorded when it is unknown, rather than assigning a cause to weak staining (standard IHC practice).
Should DDB1 staining appear in the cytoplasm or nucleus?
Assess both compartments: DDB1 is annotated in the cytoplasm and nucleus, with predominantly cytoplasmic localisation reported under some conditions (UniProt Q16531 localisation). Nuclear accumulation after UV irradiation is reported, so nuclear staining alone does not establish an IHC artefact (UniProt Q16531 localisation). HPA tissue IHC describes ubiquitous cytoplasmic expression, whereas HPA cell imaging reports enhanced nucleoplasmic localisation (HPA tissue IHC; HPA subcellular). Score cytoplasmic and nuclear staining separately on intact cells, using the same criteria across sections (standard IHC practice). Treat a change in compartment distribution as a finding to verify with controls and experimental context, not as proof of DNA damage (UniProt Q16531 function; standard IHC practice).
Could isoforms or epitope accessibility explain inconsistent DDB1 staining?
DDB1 has 2 annotated isoforms, but the supplied antibody caption does not identify its epitope or establish isoform-specific recognition (UniProt Q16531 isoforms; datasheet PB9578). It has no annotated transmembrane segment, so interpret the assay as staining an intracellular protein rather than a membrane-facing target (UniProt Q16531 topology and localisation). Annotated modifications include acetylation at residue 2 and 1067 and phosphorylation at 1125; their effects on this antibody’s IHC signal are unknown (UniProt Q16531 modified residues; datasheet PB9578). Compare serial sections under matched retrieval and detection conditions before attributing patchy staining to an isoform or modification (standard IHC practice). Document the antibody identity and any subsequently verified epitope when interpreting discrepancies (standard IHC practice).
How can IF help check the DDB1 pattern seen by IHC?
Use IF as a separate assay to examine whether DDB1 signal falls in the cytoplasm or nucleoplasm, both reported locations (UniProt Q16531 localisation; HPA subcellular). Multiplex with a marker for the cell type being evaluated and a nuclear counterstain so cell identity and compartment boundaries can be judged together (standard IF practice). Choose a fluorophore channel with low tissue autofluorescence and include single-label controls to assess bleed-through (standard IF practice). Because DDB1 is intracellular and has no transmembrane segment, permeabilise sufficiently to reach cytoplasmic and nuclear epitopes, then check that cellular structure remains interpretable (UniProt Q16531 topology and localisation; standard IF practice). Do not transfer the paraffin-section retrieval condition to IF without testing it in that assay (datasheet PB9578; standard IF practice).
What should I check when DDB1 IHC shows diffuse background?
Start with a no-primary control to identify signal from the secondary reagent, biotin-based detection, or chromogen rather than antibody-dependent staining (standard IHC practice). The selected assay used 10% goat serum blocking, a biotinylated secondary antibody, a streptavidin–biotin complex, and DAB (datasheet PB9578). Include an appropriate endogenous biotin control when interpreting this detection system, and use a peroxidase block as a general chromogenic IHC step (standard IHC practice). Compare background in tissue edges, damaged areas, and intact cell interiors before adjusting antibody concentration or wash conditions (standard IHC practice). Preserve the same detection settings across test sections so changes in background can be attributed to the step being tested (standard IHC practice).
How should I quantify DDB1 across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored cell population and assess nuclear and cytoplasmic staining separately because both DDB1 compartments are reported (UniProt Q16531 localisation). An H-score can combine the percentage of cells at each intensity, while percentage-positive cells or positive-cell density per mm² may suit other study questions (standard IHC practice). Keep intensity thresholds, counterstain, section selection, and imaging settings consistent across the comparison (standard IHC practice). Normalise cell counts to the number of evaluable cells in the defined population, or normalise density to the measured viable tissue area (standard IHC practice). Report excluded edge or necrotic regions and the antibody and retrieval conditions so a compartment shift is not mistaken for an abundance change (datasheet PB9578; standard IHC practice).
How do I distinguish a DDB1-positive pattern from artefact?
Look for staining within intact cells and assess cytoplasmic and nuclear signal against the documented DDB1 localisations (UniProt Q16531 localisation; HPA tissue IHC; HPA subcellular). HPA reports high staining in several glandular and neuronal cell populations, but also notes low consistency between antibody staining and RNA expression data (HPA tissue IHC). Investigate signal concentrated at section edges, in necrotic areas, or in a no-primary control before calling cells positive (standard IHC practice). Check endogenous peroxidase and biotin-related background when using the selected DAB and streptavidin–biotin workflow (datasheet PB9578; standard IHC practice). Interpret a nuclear shift in experimental context; DDB1 nuclear translocation is reported after UV irradiation, but staining alone does not demonstrate that mechanism (UniProt Q16531 localisation; standard IHC practice).
Boster reagents
Best DDB1 / DNA damage-binding protein 1 IHC Antibodies
PB9578 has IHC images from mouse and rat liver and human intestinal cancer tissue, plus IF data from A431 cells (PB9578 image captions). Both antibodies list human, mouse and rat reactivity (catalog).
Real IHC data
Anti-DDB1 Antibody ®
Cat # PB9578
PB9578 will render with its own mouse liver paraffin-section IHC image; its additional captions show rat liver and human intestinal cancer tissue IHC and A431 cell IF (PB9578 image captions). M00333 will render with listed IHC and IF/ICC applications and human, mouse and rat reactivity, but no supplied image validation (M00333 catalog entry).
Which to pick: Choose PB9578 for tissue IHC: its own caption documents citrate pH 6 retrieval for 20 minutes and 1 μg/ml antibody on a mouse liver paraffin section; the fixative is unreported (PB9578 IHC caption). For IF/ICC, PB9578 has an A431 cell IF image, while M00333 is a rabbit monoclonal option with IF/ICC listed but no supplied image (PB9578 IF caption; M00333 catalog entry). For cross-species tissue work, PB9578 has IHC captions for mouse, rat and human samples; both SKUs list reactivity with all three species (PB9578 IHC captions; catalog reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q16531 (DDB1_HUMAN, DNA damage-binding protein 1).
- Human Protein Atlas. DDB1 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. DDB1 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the mid piece, principal piece and end piece..
- Human Protein Atlas. DDB1 antibody validation summary (3 antibodies).
- DDB1 Regulates Sertoli Cell Proliferation and Testis Cord Remodeling by TGFβ Pathway. Genes 2019 — PMC6947845.
- Targeting CRL4 suppresses chemoresistant ovarian cancer growth by inducing mitophagy. Signal transduction and targeted therapy 2022 — PMC9731993.
- Hepatitis B virus regulatory HBx protein binding to DDB1 is required but is not sufficient for maximal HBV replication. Virology 2012 — PMC3294142.
- Role of Damage DNA-Binding Protein 1 in Pancreatic Cancer Progression and Chemoresistance. Cancers 2019 — PMC6966444.
- PubMed PMID:8530102 — UniProt-cited evidence.
- PubMed PMID:7815490 — UniProt-cited evidence.
- PubMed PMID:8538642 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for DDB1 (DNA damage-binding protein 1). Expected localisation Tissue: cytoplasmic; molecular: cytoplasm and nucleus (HPA tissue IHC; UniProt). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet PB9578). Validated IHC antibody: PB9578 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Smooth muscle. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded Mouse Liver tissue; fixative not specified (datasheet PB9578); 2. Fixation: Image fixative and duration unreported (datasheet PB9578); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: Citrate pH 6 , 20 min (datasheet PB9578); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9578); 8. Primary antibody: Rabbit anti-DDB1, 0.5-1μg/ml (datasheet PB9578); 9. Primary incubation: Overnight at 4 °C (datasheet PB9578). Watch for: Fixation: Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific); Caveat: IHC staining has low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope differences unreported (UniProt); Regulation: UV drives nuclear accumulation (UniProt). Evidence: UniProt Q16531, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet PB9578, PMC6947845, PMC9731993. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/DDB1/immunohistochemistry-ihc-guide.