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DDB1 / DNA damage-binding protein 1 · IHC design guide
Design Immunohistochemistry for DDB1
Plan DDB1 chromogenic IHC in paraffin sections using the broad cytoplasmic tissue pattern as a reference (HPA tissue IHC). The catalog antibody has an IHC-P starting range of 0.5–1 µg/mL (datasheet: PB9578).
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 | Cytoplasmic in tissue (HPA tissue IHC); nuclear after UV (UniProt) | |
| Staining pattern | Broad cytoplasmic staining across tissue cells (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 consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining has low agreement with RNA levels (HPA tissue IHC) | |
| Regulation | No specific expression induction annotated (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage is unreported (UniProt) |
Section 1
Recommended DDB1 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: PB9578) is accompanied by three published DDB1 IHC methods using mouse testes, ovarian tumors, and mouse liver (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 noteStart with citrate pH 6 heat retrieval for the catalog antibody (datasheet: PB9578); the mouse liver method specifies retrieval at 121 °C for 5 min (PMC3294142).
Section 2
What Is the Expected DDB1 Staining Pattern?
DDB1 is primarily cytoplasmic but can also localize to the nucleus (UniProt Q16531). In tissue IHC, expect broad cytoplasmic staining, including high staining in glandular cells and selected neurons (HPA: ubiquitous cytoplasmic expression; HPA: High in glandular and neuronal cells). DDB1 has no transmembrane segment (UniProt Q16531 topology). HPA rates its tissue IHC pattern Approved, while noting low consistency between antibody staining and RNA expression (HPA: Approved; low consistency).
| Cytoplasmic staining is clear in adrenal, appendix, breast, or colon glandular cells; nearby structures are distinguishable. | This matches the reported tissue pattern (HPA: High in those glandular cells; ubiquitous cytoplasmic expression). Judge the stained cell type and compartment together. An HPA High result supports an expected positive tissue, but intensity still depends on the local IHC workflow (standard IHC practice). |
| Nuclear staining accompanies cytoplasmic staining, or a sample shows predominantly nuclear signal. | Nuclear DDB1 is biologically plausible: UniProt lists both compartments and reports nuclear translocation after UV irradiation (UniProt Q16531). A predominantly nuclear result differs from HPA's broad tissue IHC description, so compare cellular morphology and controls before calling it an artefact (HPA: ubiquitous cytoplasmic expression; standard IHC practice). |
| A sharp cell-surface rim or extracellular deposit dominates instead of intracellular staining. | That compartment is discordant with reported cytoplasmic and nuclear localization and the absence of a transmembrane segment (HPA: tissue IHC profile; UniProt Q16531 topology). Treat it as a possible staining artefact and assess the detection control and tissue morphology (standard IHC practice). |
| Strong signal appears in smooth muscle cells, while expected positive cells stain weakly or inconsistently. | Smooth muscle cells are reported as Not detected (HPA: smooth muscle cells). Unexpected signal can reflect cross-reactivity or endogenous detection activity; its location alone cannot identify the cause (standard IHC practice). Check whether the same pattern appears when primary antibody is omitted. |
| Diffuse colour covers cells and surrounding tissue, or known-positive cells have no discernible signal. | Diffuse staining limits compartment scoring and can arise from nonspecific detection or incomplete removal of reagents (standard IHC practice). No signal in an HPA High tissue warrants a workflow check, but the HPA rating does not guarantee every antibody or section will stain (HPA: High examples; Approved with low RNA consistency). |
💡Expected DDB1 appearanceA convincing positive shows discernible cytoplasmic staining in the expected cell population, potentially with nuclear signal (HPA: ubiquitous cytoplasmic expression; UniProt Q16531 localization); strong glandular or selected neuronal staining fits HPA High observations, while a dominant membrane rim or diffuse deposit is suspect (HPA: tissue IHC profile; UniProt Q16531 topology).
| Tissue and cell selection | Adrenal, appendix, breast, and colon glandular cells are High; caudate and cerebral cortex neuronal cells and cerebellar Purkinje cells are also High (HPA: tissue IHC). Thyroid and epididymal glandular cells are Low, so they offer a less clear expected positive (HPA: tissue IHC). |
| Compartment and biological context | UniProt describes DDB1 as primarily cytoplasmic and capable of nuclear accumulation after UV irradiation (UniProt Q16531). HPA's tissue IHC summary is cytoplasmic; these observations should guide interpretation without making all nuclear staining erroneous (HPA: tissue IHC profile). |
| Antibody evidence | HPA045174 and CAB032821 have Approved IHC annotations; HPA068456 has no IHC annotation in the supplied record (HPA: antibody validation). The tissue profile carries an Approved rating with low staining–RNA consistency, so evaluate the actual stained cell types (HPA: tissue IHC reliability). |
| IF/ICC Q&A: what localization should be expected? | HPA reports enhanced nucleoplasmic localization in ICC-IF, with additional localization in the sperm mid, principal, and end pieces (HPA: subcellular ICC-IF). This is a separate imaging observation from HPA's cytoplasmic tissue IHC summary (HPA: tissue IHC profile). |
| Situation | Likely cause | Next action |
|---|---|---|
| No stain in an adrenal, appendix, breast, or colon glandular-cell section. | These cells are reported High, so absent signal raises a technical concern; the record does not identify a DDB1-specific fixation effect (HPA: tissue IHC; supplied source scope). | Check tissue morphology, primary-antibody application, detection reagents, and a positive control in the same run (standard IHC practice). If retrieval is adjusted, treat it as general workflow optimization, not a documented DDB1 fixation requirement. |
| Only nuclei stain in an otherwise readable tissue section. | Nuclear DDB1 is possible, although HPA describes ubiquitous cytoplasmic tissue staining (UniProt Q16531 localization; HPA: tissue IHC profile). | Inspect whether signal follows intact nuclei and appears in expected cells; compare another validated section or antibody result when available (standard IHC practice). Record the compartment difference instead of automatically rejecting the result. |
| Smooth muscle cells show prominent chromogen. | This conflicts with HPA's Not detected entry and may reflect off-target antibody binding or endogenous detection activity (HPA: smooth muscle cells; standard IHC practice). | Run a primary-omission control and review the detection blocking steps; compare the smooth-muscle signal with expected positive cells on the same slide (standard IHC practice). |
| Brown colour is widespread, with no clear cellular boundaries. | The pattern cannot establish DDB1's reported cytoplasmic distribution and may represent nonspecific background (HPA: tissue IHC profile; standard IHC practice). | Review washing, blocking, antibody concentration, and detection development against control sections; score localization only where cell boundaries remain interpretable (standard IHC practice). |
| A sharp surface rim is the main staining pattern. | DDB1 is reported in cytoplasm and nucleus and lacks a transmembrane segment (UniProt Q16531 localization and topology). A dominant rim therefore needs scrutiny. | Compare with a primary-omission control and assess whether deposits track section edges or damaged areas (standard IHC practice). Do not count an isolated rim as the expected intracellular pattern. |
| Low-level or variable staining appears in thyroid, epididymis, placenta, or adipose tissue. | HPA reports Low staining in thyroid and epididymal glandular cells, placental decidual cells, and adipocytes (HPA: tissue IHC). These are weak comparison tissues. | Use an HPA High cell population to assess assay performance, then score these tissues by cell type and compartment; avoid calling variable low signal a failed run by itself (HPA: tissue IHC; standard IHC 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 adrenal gland first and confirm staining in its glandular cells (HPA: High in adrenal gland glandular cells). Run smooth muscle as the tissue negative (HPA: Not detected in smooth muscle cells); on the adrenal slide, treat non-glandular cells as internal negative controls only where they show no specific staining, since their DDB1 status is not given by the supplied HPA 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 control and a matched nonimmune rabbit IgG isotype control (selected PB9578 caption: rabbit primary antibody); use DDB1 knockout tissue as a biological specificity control if available. Quench endogenous peroxidase and check for endogenous biotin signal before using the caption’s streptavidin–biotin/DAB detection system (selected PB9578 caption: SABC with DAB).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported; the selected PB9578 paraffin-section caption does not state a fixative (selected PB9578 caption: fixative unreported). The reported IHC workflow uses heat retrieval in citrate buffer at pH 6 for 20 minutes, but its necessity across specimens is unreported (selected PB9578 caption: heat-mediated citrate retrieval). Neither frozen sections nor IF are established as easier by the supplied evidence; in adrenal gland, assess any endogenous peroxidase or biotin background before interpreting DAB staining (selected PB9578 caption: SABC with DAB).
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
Troubleshoot DDB1 staining in paraffin sections by checking retrieval, compartment patterns, controls and scoring before interpreting biological differences.
How should I adjust retrieval when DDB1 staining is weak?
Start with heat-mediated citrate retrieval at pH 6 for 20 minutes (datasheet PB9578). The selected paraffin-section example used that condition before overnight incubation with 1 μg/mL primary antibody at 4°C (caption PB9578). If staining remains weak, compare a second retrieval condition on adjacent sections while holding antibody concentration and detection constant (standard IHC practice). Include a previously staining section in each run so a change in retrieval can be distinguished from a failed detection run (standard IHC practice). Record nuclear and cytoplasmic staining separately because both compartments are biologically plausible for DDB1 (UniProt Q16531 subcellular location).
Could fixation explain inconsistent DDB1 staining between paraffin blocks?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative (caption PB9578). Compare blocks with documented fixation histories using the same citrate pH 6, 20-minute retrieval and the same staining run (datasheet PB9578; standard IHC practice). If histories differ, interpret intensity differences cautiously and check morphology, tissue preservation and background before changing antibody concentration (standard IHC practice). Keep a reference block in subsequent runs to assess whether staining varies with block processing or the assay (standard IHC practice). DDB1 localisation or modified residues alone cannot establish how its epitope responds to fixation (UniProt Q16531 subcellular location and modified residues).
Is cytoplasmic DDB1 staining credible when I expected nuclear signal?
Cytoplasmic staining is plausible: DDB1 is described as primarily cytoplasmic and able to translocate to the nucleus after UV irradiation (UniProt Q16531 subcellular location). HPA tissue IHC reports ubiquitous cytoplasmic expression, whereas its subcellular imaging reports enhanced nucleoplasmic localisation (HPA tissue IHC; HPA subcellular). Score the nuclear and cytoplasmic compartments independently, using the counterstain to define nuclei and adjacent cells to assess background (standard IHC practice). Compare like tissue regions and processing conditions before calling a compartment shift (standard IHC practice). A localisation change in a section alone does not establish UV exposure or DNA damage (UniProt Q16531 subcellular location; standard IHC interpretation).
Could an unrecognised DDB1 isoform or epitope cause weak staining?
DDB1 has 2 annotated isoforms, but the supplied antibody evidence does not map the recognised epitope to either isoform (UniProt Q16531 isoforms; caption PB9578). Its annotated chain spans residues 2–1140, and modified residues include positions 2, 1067 and 1125 (UniProt Q16531 processing and modified residues). Do not assign a staining difference to isoform usage or modification without epitope mapping and independent evidence (standard IHC interpretation). First compare adjacent sections under the documented citrate pH 6 retrieval condition and a consistent detection run (datasheet PB9578; standard IHC practice). If the antibody's immunogen becomes available, check whether its sequence is shared by the isoforms (standard antibody validation practice).
How can IF help assess a disputed DDB1 compartment pattern?
Use IF as an independent localisation check and multiplex DDB1 with a marker for the expected cell type in the section (standard IF practice). Choose fluorophores after inspecting tissue autofluorescence, favouring a detection channel with clear signal separation and including single-colour controls (standard IF practice). DDB1 has no annotated transmembrane segment, so optimise permeabilisation for access to its cytoplasmic and nuclear epitopes, then check that nuclear morphology remains intact (UniProt Q16531 topology and subcellular location; standard IF practice). HPA reports enhanced nucleoplasmic localisation in subcellular imaging, while its tissue IHC profile is cytoplasmic (HPA subcellular; HPA tissue IHC). Report agreement or disagreement by compartment without treating IF images as validation of this catalog antibody in tissue sections (standard antibody validation practice).
What should I check when DDB1 staining appears diffusely brown?
The selected assay uses a biotinylated secondary, an avidin–biotin detection complex and DAB, so examine a no-primary control for detection-related background (caption PB9578; standard IHC practice). Include a peroxidase block and compare an avidin–biotin blocking step if the tissue shows background with that detection chemistry (standard chromogenic IHC practice). The caption used 10% goat serum blocking and 1 μg/mL primary antibody overnight at 4°C; keep these documented conditions as the starting reference (caption PB9578). Inspect vessel contents, damaged edges and poorly preserved areas before accepting diffuse colour as cellular signal (standard IHC practice). Genuine DDB1 staining may include cytoplasm, so background assessment must consider cellular boundaries and control sections (UniProt Q16531 subcellular location; standard IHC practice).
How should I score DDB1 across sections with different staining patterns? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue region and eligible cell population before scoring, then record nuclear and cytoplasmic positivity separately (standard IHC quantification; UniProt Q16531 subcellular location). For each compartment, report the percentage of positive cells and an H-score from 0 to 300 when intensity categories are reproducible (standard IHC quantification). If counting spatially restricted positive cells, report density per mm² of evaluable tissue and exclude folds, necrosis and section edges (standard IHC quantification). Normalise cell counts to the number of eligible cells, or area-based counts to evaluable tissue area, using the same threshold across batches (standard IHC quantification). HPA reports low consistency between antibody staining and RNA expression, so avoid using RNA abundance as the sole scoring benchmark (HPA tissue IHC reliability).
How do I distinguish true DDB1 positivity from staining artefact?
Accept a DDB1 pattern only when colour follows intact cells, repeats in comparable regions and exceeds the appropriate no-primary control (standard IHC interpretation). Cytoplasmic signal is compatible with the HPA tissue profile, while nuclear signal is biologically possible given DDB1 localisation and damage-associated nuclear translocation (HPA tissue IHC; UniProt Q16531 subcellular location). Check unexpected cell staining against tissue morphology; HPA lists high glandular-cell staining in colon and no detected staining in smooth muscle cells (HPA tissue IHC). Exclude edge accentuation, necrotic material and endogenous peroxidase signal before scoring DAB deposits (standard chromogenic IHC practice). A positive section cannot by itself demonstrate DNA repair activity or recruitment to damage sites (UniProt Q16531 function; standard IHC interpretation).
Boster reagents
Best DDB1 / DNA damage-binding protein 1 IHC Antibodies
Anti-DDB1 antibodies are listed for IHC and IF/ICC in human, mouse, and rat; PB9578 has pictured paraffin-section IHC across all three species and IF in A431 cells (catalog; PB9578 image captions).
Real IHC data
Anti-DDB1 Antibody ®
Cat # PB9578
PB9578 has IHC images from paraffin sections of mouse and rat liver and human intestinal cancer, plus an IF image from A431 cells (PB9578 image captions). M00333 lists IHC and IF/ICC applications and human, mouse, and rat reactivity, but has no supplied IHC or IF image captions (M00333 catalog).
Which to pick: For tissue IHC, choose PB9578: its captions document paraffin sections with citrate retrieval, though the fixative is unreported (PB9578 IHC image captions). For IF/ICC, PB9578 has pictured IF in A431 cells; rabbit monoclonal M00333 lists IF/ICC but has no supplied image caption (PB9578 IF image caption; M00333 catalog). For cross-species IHC, both list human, mouse, and rat reactivity, while PB9578 has pictured IHC examples from each species (catalog reactivity; PB9578 IHC image captions).
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 Cytoplasmic in tissue (HPA tissue IHC); nuclear after UV (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 consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Staining has low agreement with RNA levels (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage is unreported (UniProt); Regulation: No specific expression induction annotated (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.