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XRCC4 / DNA repair protein XRCC4 · IHC design guide
Design Immunohistochemistry for XRCC4
Plan XRCC4 IHC in paraffin sections around the ubiquitous nuclear staining reported in tissue (HPA tissue IHC). The guide covers controls, chromogenic detection and scoring, with an IHC-validated antibody starting range of 2–5 μg/ml (datasheet: A00787-2).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
XRCC4 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 | Nuclear tissue staining (HPA tissue IHC) | |
| Staining pattern | Widespread nuclear staining across tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A00787-2) | |
| Positive control | Adipose tissue+4 more · see all | |
| Negative control | Liver+2 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Antibody staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Widely expressed (UniProt) | |
| Isoform / epitope | 3 isoforms and a 266–336 C-terminal chain; check epitope (UniProt) |
Section 1
Recommended XRCC4 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published XRCC4 IHC methods for hepatocellular carcinoma tissue (PMC5675685) and breast cancer paraffin sections (PMC7661505).
| Sample | Paraffin-embedded human lung squamous cell carcinoma tissue; fixative not specified (datasheet A00787-2) |
| Fixation | Image fixative and duration unreported (datasheet A00787-2); 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 A00787-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A00787-2) |
| Primary antibody | Rabbit anti-XRCC4, 2-5 μg/ml (datasheet A00787-2) |
| Primary incubation | Overnight at 4 °C (datasheet A00787-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A00787-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | XRCC4-positive staining in adipocytes of adipose tissue (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet A00787-2); the breast cancer study used EDTA at pH 9.0 (PMC7661505 methods).
Section 2
What Is the Expected XRCC4 Staining Pattern?
XRCC4 should appear predominantly in nuclei across many cell types: HPA describes ubiquitous nuclear tissue staining with Supported reliability and medium consistency with RNA data (HPA tissue IHC). UniProt also places XRCC4 in the nucleus, on chromosomes and in the cytoplasm, including at double-strand breaks; it has no transmembrane segment (UniProt Q13426). Interpret paraffin-section staining against the expected nuclear pattern and the specific cell types reported by HPA.
| Distinct nuclear chromogen in adipocytes, bone-marrow hematopoietic cells or bronchial respiratory epithelial cells. | This matches reported High staining in those cells and HPA's ubiquitous nuclear profile (HPA tissue IHC). Assess the named cells: a positive field alone does not show that staining occupies the expected compartment. |
| Predominantly cytoplasmic staining, with little or no nuclear staining, in an otherwise positive-looking section. | Question antibody specificity or slide background before calling this the expected IHC result (HPA tissue IHC: ubiquitous nuclear expression). UniProt lists cytoplasm as a location, so an isolated cytoplasmic signal is not, by itself, proof of an artefact (UniProt Q13426). |
| Strong staining in cholangiocytes, ovarian stromal cells or smooth-muscle cells. | These named cell populations were Not detected by HPA tissue IHC; their signal merits a cross-reactivity or endogenous detection-activity check (HPA tissue IHC; general IHC practice). The finding does not make every cell in liver, ovary or smooth-muscle-containing tissue negative. |
| Diffuse chromogen obscures nuclear boundaries or appears across cells and surrounding tissue. | The distribution is difficult to score against HPA's nuclear profile (HPA tissue IHC). Review a no-primary control and detection-system background; nonspecific staining or endogenous activity are general IHC possibilities, not established XRCC4 properties (general IHC practice). |
| No nuclear signal in a section containing well-preserved, expected-positive cells. | First confirm that the scored cells match an HPA High population, such as bone-marrow hematopoietic cells (HPA tissue IHC). An absent signal calls for assay and tissue-quality checks; it does not alone establish biological absence (general IHC practice). |
💡Expected XRCC4 appearanceCall a convincing positive result when the chromogen is clearly nuclear in the relevant cells, with strong staining plausible in HPA High populations; predominant nonnuclear staining or signal in HPA Not detected cell populations is a finding to investigate (HPA tissue IHC; UniProt Q13426).
| Cell type and compartment | HPA reports ubiquitous nuclear expression but gives cell-specific levels: adipocytes and bone-marrow hematopoietic cells are High, while cholangiocytes are Not detected (HPA tissue IHC). Score the identified cells and their nuclei, rather than assigning one result to a whole tissue. |
| Strength of the tissue evidence | The tissue profile is Supported, with medium consistency between antibody staining and RNA expression (HPA tissue IHC). Use its named positive and undetected populations as interpretation references, while treating a disagreement as a reason to verify the assay and cell identity. |
| Antibody-specific validation | HPA006801 is IHC Supported; HPA051538 has no listed IHC status in the supplied record (HPA antibody validation). ICC Enhanced for both antibodies concerns ICC evidence and does not confer IHC validation on the second antibody (HPA antibody validation). |
| Isoforms and epitope coverage | UniProt lists 3 XRCC4 isoforms and a C-terminal chain spanning residues 266–336 (UniProt Q13426). The supplied evidence gives no antibody epitope, so it cannot establish which forms a given antibody detects or predict a different tissue stain. |
| Antigen retrieval and processing | Use the IHC-validated antibody's established paraffin-section conditions when available, and document retrieval during optimisation (general IHC practice). No target-specific fixation sensitivity or retrieval dependence is supplied here; UniProt's processing and modification annotations do not establish either (UniProt Q13426). |
| IF/ICC Q&A: Where should XRCC4 appear? | Predominantly in the nucleoplasm: HPA calls it an enhanced main location and lists ICC-IF images from A-431, U-251MG and U2OS (HPA subcellular ICC-IF). That observation supports localisation interpretation, without specifying an IF protocol for this IHC guide. |
| Situation | Likely cause | Next action |
|---|---|---|
| No stain in expected-positive hematopoietic cells. | The field may lack the named cells, or the IHC assay may have failed; HPA reports them as High (HPA tissue IHC; general IHC practice). | Confirm cell identity and tissue preservation, then check the run's positive control and documented antibody, retrieval and detection conditions (general IHC practice). Do not infer XRCC4 loss from one blank section. |
| Chromogen is mainly cytoplasmic. | The pattern departs from HPA's ubiquitous nuclear tissue profile, although UniProt also lists cytoplasm (HPA tissue IHC; UniProt Q13426). Background or antibody-dependent staining remains possible (general IHC practice). | Compare nuclear and cytoplasmic signal in an HPA High cell population, inspect the no-primary control, and report the observed compartment rather than forcing a nuclear-positive call (HPA tissue IHC; general IHC practice). |
| Named HPA-undetected cells stain strongly. | Cholangiocytes, ovarian stromal cells and smooth-muscle cells are Not detected in the supplied HPA tissue profile (HPA tissue IHC). Cross-reactivity or endogenous detection activity is possible (general IHC practice). | Recheck cell identification and the no-primary control; compare staining in an HPA High population on the same run when available (HPA tissue IHC; general IHC practice). |
| Background makes nuclear scoring uncertain. | Diffuse chromogen can obscure the compartment needed to assess HPA's nuclear profile (HPA tissue IHC). Nonspecific primary or detection-system staining can contribute (general IHC practice). | Inspect no-primary and other appropriate run controls, then review blocking, antibody concentration and detection conditions under the established IHC workflow (general IHC practice). Score only nuclei whose boundaries remain interpretable. |
| Different tissue areas give different apparent results. | HPA's broadly nuclear summary coexists with cell-specific High and Not detected entries (HPA tissue IHC). A change in sampled cell population can therefore change the field-level impression. | Record the tissue and cell population for each scored area; compare like cells across fields and note any limited or damaged material (HPA tissue IHC; general IHC practice). Avoid treating an undetected cell type as a whole-tissue negative. |
| An antibody with ICC images gives an uncertain paraffin-section result. | ICC and IHC have separate validation entries: HPA051538 is ICC Enhanced but has no listed IHC status, whereas HPA006801 is IHC Supported (HPA antibody validation). | Check the exact antibody's IHC status and use an IHC-validated antibody with appropriate tissue and run controls for a paraffin-section conclusion (HPA antibody validation; general IHC practice). |
Experiment evidence
IHC & IF images showing XRCC4 staining
A00787-2 · IHCIHC analysis of XRCC4 using anti-XRCC4 antibody (A00787-2). XRCC4 was detected in a paraffin-embedded section of human lung squamous cell ca
A00787-2 · IHCIHC analysis of XRCC4 using anti-XRCC4 antibody (A00787-2). XRCC4 was detected in a paraffin-embedded section of human breast duct carcinoma
A00787-2 · IFIF analysis of XRCC4 using anti-XRCC4 antibody (A00787-2). XRCC4 was detected in an immunocytochemical section of U20S cells. Enzyme antigen
PB9908 · IHCIHC analysis of XRCC4 using anti-XRCC4 antibody (PB9908). XRCC4 was detected in a paraffin-embedded section of human intestinal cancer tissu
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000152422-XRCC4/tissue/cerebral+cortex#imid_2119584
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000152422-XRCC4/tissue/colon#imid_2119526
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000152422-XRCC4/tissue/liver#imid_2119511
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000152422-XRCC4/tissue/kidney#imid_2119520
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA051538; green = XRCC4, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA051538; green = XRCC4, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for XRCC4 IHC & IF
🧪Run breast first: glandular cells should stain (HPA: High in breast glandular cells). Use liver cholangiocytes as the negative tissue (HPA: Not detected in liver cholangiocytes); on the breast slide, any cells without specific XRCC4 signal should show counterstain alone, but their absence of signal does not establish that they are biological negatives (UniProt Q13426: widely expressed).
Positive control tissue: Adipose tissue (Adipocytes, HPA High)
Negative control tissue: Liver (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show XRCC4 in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; a concentration-matched nonimmune rabbit IgG control matched to the primary antibody’s clonality once confirmed (caption: rabbit anti-XRCC4; clonality unreported); and XRCC4 knockout tissue or a validated immunizing-peptide competition control. For chromogenic staining, quench endogenous peroxidase and check the breast section for background before scoring glandular cells (caption: peroxidase secondary and DAB detection; HPA: High in breast glandular cells).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A00787-2 paraffin-section caption does not state the fixative (caption: fixative unreported). Heat retrieval with EDTA at pH 8.0 is documented for that paraffin IHC example, but retrieval dependency and whether frozen sections or IF are easier are unreported (caption: heat-mediated EDTA retrieval; HPA: ICC-IF images available for A-431, U-251MG and U2OS). In breast sections, endogenous peroxidase can produce chromogenic background, so interpret glandular staining against the no-primary control (caption: peroxidase secondary and DAB detection; HPA: High in breast glandular cells).
HPA tissue IHC evidence for XRCC4
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — 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 |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | High | Protein (IHC) | HPA → |
| 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 → |
Section 3
Advanced XRCC4 IHC Tips
Troubleshoot XRCC4 staining in paraffin section IHC using the catalog antibody’s tissue image, reported localisation, and tissue expression as reference points.
Which retrieval condition should I try first for weak XRCC4 staining?
Start with heat mediated antigen retrieval in EDTA at pH 8.0 (datasheet A00787-2). That condition preceded XRCC4 staining in a paraffin section of human lung squamous cell carcinoma with the catalog antibody (datasheet A00787-2). If nuclear signal remains weak, check heating consistency and section adhesion before comparing a fallback retrieval buffer on adjacent sections (standard IHC practice). Keep primary antibody concentration and chromogen development comparable across those sections so retrieval is the variable being assessed (standard IHC practice). The reported image used 2 μg/ml primary antibody overnight at 4°C, providing a documented starting point for that comparison (datasheet A00787-2).
How should I troubleshoot fixation related loss of XRCC4 signal?
Target specific fixation sensitivity is unknown: the selected paraffin section caption does not state a fixative (datasheet A00787-2). Record each specimen’s fixative, fixation duration, processing history, and section age before attributing weak nuclear staining to fixation (standard IHC practice). Compare sections with known handling histories under the same EDTA pH 8.0 retrieval and 2 μg/ml primary conditions documented for the catalog antibody (datasheet A00787-2). If staining differs, repeat with matched positive tissue and negative controls, then adjust one processing variable at a time (standard IHC practice). Nuclear localisation and XRCC4 phosphorylation do not establish how this epitope responds to fixation (UniProt Q13426 localisation and modified residues).
How should I evaluate nuclear versus cytoplasmic XRCC4 staining?
Prioritise interpretable nuclear staining because tissue IHC shows ubiquitous nuclear expression, and cell imaging places XRCC4 in the nucleoplasm (HPA tissue IHC; HPA subcellular). XRCC4 is also annotated in the cytoplasm and at chromosomes, with recruitment to double strand break sites (UniProt Q13426 subcellular location). Assess nuclear and cytoplasmic compartments separately against the counterstain rather than merging them into one positive score (standard IHC practice). If diffuse cytoplasmic DAB dominates, inspect no primary controls and the distribution of staining across section edges before assigning it to XRCC4 (standard IHC practice). Treat focal nuclear enrichment as a localisation observation, not proof of repair activity, without independent damage evidence (UniProt Q13426 function; standard IHC practice).
Could isoforms or modifications explain inconsistent XRCC4 staining?
XRCC4 has 3 annotated isoforms and a reported C terminal product spanning residues 266–336 (UniProt Q13426 isoforms and processing). Its listed modifications include phosphorylation at residues 53, 193, 229, 232, and 233 (UniProt Q13426 modified residues). The supplied IHC caption does not identify the catalog antibody’s epitope or demonstrate isoform specific recognition (datasheet A00787-2). If samples stain differently, request epitope mapping and compare staining with an independently validated antibody or orthogonal assay before proposing isoform or modification dependence (standard IHC practice). Keep retrieval and detection conditions matched during that comparison, since processing differences can also alter apparent staining (standard IHC practice).
How can I check XRCC4 localisation by IF alongside this IHC result?
Use IF as a separate localisation check, comparing nuclear signal with the chromogenic IHC pattern rather than assuming identical antibody performance (HPA tissue IHC; standard IHC/IF practice). For bronchial tissue, multiplex with a respiratory epithelial cell marker to identify a cell population reported to stain highly for XRCC4 (HPA: High in bronchus respiratory epithelial cells). Choose spectrally separated fluorophores and favour a channel with low measured tissue autofluorescence; include single colour and no primary controls (standard IF practice). Because XRCC4 has no transmembrane segment and is found in the nucleoplasm, permeabilisation must permit antibody access across cellular and nuclear barriers (UniProt Q13426 topology; HPA subcellular). Optimise that step for the antibody’s epitope without transferring an unreported IF fixation condition from the paraffin image (datasheet A00787-2; standard IF practice).
What should I check when XRCC4 DAB staining is widespread?
First compare the DAB pattern with a no primary control and inspect whether signal follows tissue folds, section edges, or damaged areas (standard IHC practice). The documented paraffin image used 10% goat serum block, a peroxidase conjugated secondary, and DAB development (datasheet A00787-2). Include a peroxidase blocking step and check for residual endogenous enzyme activity before calling diffuse brown staining XRCC4 positive (standard chromogenic IHC practice). Titrate detection and review wash stringency if background persists, keeping the documented 2 μg/ml primary condition as a reference rather than an assumed optimum (datasheet A00787-2; standard IHC practice). Give nuclear signal greater interpretive weight when controls are clean, consistent with the reported tissue pattern (HPA tissue IHC).
How should I quantify XRCC4 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and nuclear region of interest before scoring, because XRCC4 tissue IHC is described as ubiquitously nuclear (HPA tissue IHC). Report the percentage of positive nuclei and staining intensity on a prespecified 0–3 scale, or calculate a nuclear H score from 0–300 (standard IHC scoring practice). Normalise positive counts to all assessable nuclei in the same annotated tissue compartment; exclude necrosis, folds, and poorly preserved areas consistently (standard IHC practice). If reporting positive cell density per mm², specify the assessed tissue area and retain the same threshold across specimens (standard IHC scoring practice). Record background controls and batch conditions alongside scores so differences can be reviewed against staining variation (standard IHC practice).
When is apparent XRCC4 positivity likely to be artefactual?
A convincing result has cellular nuclear staining in well preserved tissue, matching the reported ubiquitous nuclear IHC profile and nucleoplasmic localisation (HPA tissue IHC; HPA subcellular). Compare the stained cell type with the annotated tissue context: bronchial respiratory epithelial cells are reported high, while liver cholangiocytes are reported not detected (HPA tissue IHC). Brown signal concentrated at section edges, in necrotic regions, or in a no primary control warrants an artefact check (standard IHC practice). Diffuse cytoplasmic staining alone needs independent confirmation, although cytoplasmic XRCC4 is annotated (UniProt Q13426 subcellular location; standard IHC practice). Positive staining indicates detectable antigen under these conditions; it cannot by itself establish active double strand break repair (UniProt Q13426 function; standard IHC practice).
Boster reagents
Best XRCC4 / DNA repair protein XRCC4 IHC Antibodies
Both anti-XRCC4 antibodies have human paraffin-section IHC images (catalog: reactivity and IHC captions); A00787-2 also has IF images of U20S cells and human colorectal adenocarcinoma sections (catalog: IF captions).
Real IHC data
Anti-XRCC4 Antibody ®
Cat # A00787-2
Real IHC data
Anti-XRCC4 Antibody ®
Cat # PB9908
A00787-2 has IHC images from human lung squamous cell carcinoma, breast duct carcinoma, colorectal adenocarcinoma and liver cancer sections, plus IF images from U20S cells and human colorectal adenocarcinoma sections (catalog: A00787-2 image captions). PB9908 has an IHC image from a human intestinal cancer section (catalog: PB9908 IHC caption).
Which to pick: For paraffin-section IHC, either SKU has an example: A00787-2 in human lung squamous cell carcinoma and PB9908 in human intestinal cancer, each using EDTA retrieval at pH 8.0 (catalog: each SKU’s own IHC caption); neither caption reports the fixative (catalog: IHC captions). For IF/ICC, choose A00787-2, which lists both applications and has IF images; PB9908 lists IHC but not IF/ICC (catalog: applications and IF captions). Neither SKU has documented cross-species reactivity, and clonality is unreported for both (catalog: reactivity and clone fields).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q13426 (XRCC4_HUMAN, DNA repair protein XRCC4).
- Human Protein Atlas. XRCC4 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. XRCC4 subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. XRCC4 antibody validation summary (2 antibodies).
- Prognostic significance of XRCC4 expression in hepatocellular carcinoma. Oncotarget 2017 — PMC5675685.
- Silencing of XRCC4 increases radiosensitivity of triple-negative breast cancer cells. Bioscience reports 2019 — PMC6423307.
- Significant value of XRCC2 and XRCC9 expression in the prognosis of human ovarian carcinoma. Journal of Cancer 2021 — PMC8425215.
- The diagnostic value of DNA repair gene in breast cancer metastasis. Scientific reports 2020 — PMC7661505.
- PubMed PMID:8548796 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for XRCC4 (DNA repair protein XRCC4). Expected localisation Nuclear tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A00787-2). Validated IHC antibody: A00787-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adipose tissue (Adipocytes); negative Liver. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung squamous cell carcinoma tissue; fixative not specified (datasheet A00787-2); 2. Fixation: Image fixative and duration unreported (datasheet A00787-2); 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 A00787-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A00787-2); 8. Primary antibody: Rabbit anti-XRCC4, 2-5 μg/ml (datasheet A00787-2); 9. Primary incubation: Overnight at 4 °C (datasheet A00787-2). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Antibody staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 3 isoforms and a 266–336 C-terminal chain; check epitope (UniProt); Regulation: Widely expressed (UniProt). Evidence: UniProt Q13426, HPA tissue IHC · Supported, HPA ICC-IF · nucleoplasm., Datasheet A00787-2, PMC5675685, PMC7661505. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/XRCC4/immunohistochemistry-ihc-guide.