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XRCC4 / DNA repair protein XRCC4 · IHC design guide
Design Immunohistochemistry for XRCC4
Plan chromogenic XRCC4 IHC in paraffin sections around a broad nuclear staining pattern (HPA tissue IHC). Start the IHC-validated antibody A00787-2 at 2–5 μg/ml (datasheet A00787-2), and score nuclear staining with matched controls.
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 staining in tissue IHC (HPA tissue IHC) | |
| Staining pattern | Broad 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 | Cholangiocytes may lack detectable staining (HPA tissue IHC) | |
| Regulation | Widely expressed (UniProt) | |
| Isoform / epitope | 3 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended XRCC4 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet A00787-2) with three published XRCC4 tissue IHC methods (PMC5675685; PMC8425215; 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 pH 8.0 retrieval (datasheet A00787-2). If needed, compare the published citrate and EDTA pH 9.0 conditions (PMC8425215; PMC7661505).
Section 2
What Is the Expected XRCC4 Staining Pattern?
XRCC4 should appear mainly in nuclei across many tissues (HPA tissue IHC: ubiquitous nuclear expression; Supported reliability). UniProt also lists chromosome and cytoplasm localization, including recruitment to double-strand breaks (UniProt Q13426: subcellular location). It has no transmembrane segment (UniProt Q13426: topology). Expect staining in documented positive cells, including bone marrow hematopoietic cells and breast glandular cells (HPA tissue IHC: High).
| Nuclear staining in many cell types, with clear signal in bone marrow hematopoietic cells. | This fits the reported ubiquitous nuclear pattern and a documented High cell population (HPA tissue IHC: profile; bone marrow, High). Score the relevant cells and nuclear compartment together; a positive-looking section alone does not establish antibody specificity. |
| Predominantly cytoplasmic, membranous, or extracellular staining with little nuclear signal. | The dominant tissue pattern should be nuclear (HPA tissue IHC: ubiquitous nuclear expression). UniProt lists cytoplasm as an additional location, so limited cytoplasmic signal is not automatically false (UniProt Q13426: subcellular location). Strong membrane or extracellular signal lacks support from the supplied localization and topology record (UniProt Q13426: no transmembrane segment). |
| Strong signal in liver cholangiocytes, ovarian stroma, or smooth muscle cells. | These populations were reported as Not detected in the supplied tissue observations (HPA tissue IHC: liver; ovary; smooth muscle). Check cell identification and controls, then consider cross-reactivity or endogenous chromogenic activity. A discrepancy with HPA is a reason to investigate, not proof that an individual cell cannot express XRCC4. |
| Broad chromogen haze covers nuclei, cytoplasm, and surrounding tissue. | A haze that obscures cell boundaries cannot be scored as the reported nuclear pattern (HPA tissue IHC: profile). In chromogenic IHC, nonspecific antibody binding, insufficient blocking or washing, and endogenous detection activity can produce background (general IHC practice); compare with a no-primary control. |
| No nuclear signal in bone marrow hematopoietic cells or breast glandular cells. | Both are listed as High in the supplied tissue observations (HPA tissue IHC: bone marrow; breast). First check that the expected cells are present and the staining run worked. HPA's overall tissue reliability is Supported with medium consistency against RNA, so a single negative section requires controls and repeat assessment (HPA tissue IHC: reliability). |
💡Expected XRCC4 appearanceCall positive when documented cell populations show interpretable nuclear chromogen signal, potentially strong in HPA High cells; dominant membrane or extracellular staining, or staining confined to HPA Not detected populations, needs investigation (HPA tissue IHC: profile and levels; UniProt Q13426: topology).
| How strong is the tissue-pattern evidence? | HPA calls the tissue IHC result Supported and describes medium consistency between staining and RNA expression (HPA tissue IHC: reliability). Its positive and Not detected entries are observations for named cell populations, not universal intensity cutoffs; score each compartment and cell type against the relevant entry. |
| Which antibody evidence applies to IHC? | HPA006801 is listed as IHC Supported; HPA051538 has no IHC validation entry in the supplied antibody record (HPA antibodies: IHC status). Both have ICC Enhanced entries (HPA antibodies: ICC status). Do not transfer an ICC validation label to an IHC result. |
| Could isoforms or modifications affect interpretation? | UniProt lists three XRCC4 isoforms and several modified residues, including phosphorylation sites (UniProt Q13426: isoforms; modified residues). The supplied record gives no antibody epitope or variant-specific staining data, so an unusual tissue pattern cannot be assigned to an isoform or modification from these sources alone. |
| Is a processed or shed extracellular pool expected? | The listed full-length chain spans residues 1–336, with a C-terminal 266–336 entry; no signal peptide, propeptide, glycosylation sites, or transmembrane segment are listed (UniProt Q13426: processing; glycosylation; topology). These annotations do not support interpreting extracellular chromogen as a normal shed XRCC4 pattern. |
| IF/ICC Q&A: where should signal appear? | In the separate IF/ICC application, HPA reports enhanced nucleoplasmic localization and images from A-431, U-251MG, and U2OS cells (HPA subcellular: location; imaged cell lines). That supports a nucleoplasmic IF expectation; it does not supply an IHC staining protocol or change the tissue IHC reliability rating. |
| Situation | Likely cause | Next action |
|---|---|---|
| A documented High population is unstained while the section appears otherwise intact. | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. | Confirm cell identity and a functioning positive control, then review retrieval, primary incubation, detection, and counterstain using the IHC-validated antibody's established IHC-P instructions (general IHC practice). |
| Signal is mostly cytoplasmic with weak nuclei. | Some cytoplasmic localization is annotated, but ubiquitous nuclear staining is the observed tissue pattern (UniProt Q13426: subcellular location; HPA tissue IHC: profile). The observed balance may reflect background or a genuine difference that needs verification. | Compare nuclear and cytoplasmic signal in documented High cells and inspect a no-primary control; repeat with an IHC-supported reagent if the compartment pattern remains discordant (HPA antibodies: HPA006801 IHC Supported; general IHC practice). |
| Membranes or extracellular material stain prominently. | That distribution lacks support from the nuclear tissue profile and the absence of a transmembrane segment (HPA tissue IHC: profile; UniProt Q13426: topology). Nonspecific binding or detection background is possible (general IHC practice). | Check a no-primary control, blocking, and washing; score only cellular signal with a defensible compartment assignment (general IHC practice). |
| Cells listed as Not detected are strongly positive. | The result differs from HPA observations for liver cholangiocytes, ovarian stroma, or smooth muscle cells (HPA tissue IHC: Not detected entries). Misidentified cells, cross-reactivity, or endogenous activity are possible (general IHC practice). | Verify cell identity and compare nearby documented positive cells, a no-primary control, and repeat sections before treating the discordance as biological (HPA tissue IHC: profile; general IHC practice). |
| The entire section has diffuse chromogen background. | Nonlocalized color can result from nonspecific binding, incomplete washing, or endogenous detection activity (general IHC practice). It cannot establish the nuclear XRCC4 pattern reported by HPA (HPA tissue IHC: profile). | Inspect the no-primary control, improve blocking and washes as appropriate to the detection system, and adjust detection exposure under the established IHC workflow (general IHC practice). |
| IF/ICC appears nucleoplasmic, but matched tissue IHC is inconclusive. | The applications have different HPA evidence: enhanced nucleoplasmic ICC localization versus Supported tissue IHC with medium RNA consistency (HPA subcellular: main location; HPA tissue IHC: reliability). Agreement in IF does not validate the tissue section. | Evaluate the IHC section using its own positive cell populations and controls; use the separate IF/ICC guide for fluorescence workflow details (HPA tissue IHC: High populations; 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 adipose tissue first and look for staining in adipocytes (HPA: High in adipocytes). Run smooth muscle as the negative tissue (HPA: Not detected in smooth muscle cells); on the adipose slide, any unstained neighboring cells should show only background signal, without assuming they are biologically XRCC4-negative.
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; concentration-matched rabbit isotype IgG; and, if available, an XRCC4-knockout specimen processed in parallel (caption: rabbit anti-XRCC4 antibody; standard IHC controls). Quench endogenous peroxidase for DAB detection and assess adipose autofluorescence separately if using IF (caption: HRP/DAB detection; standard IHC/IF practice).
⚠️Feasibility: A target-specific fixation window is unreported, and the selected A00787-2 tissue-IHC caption does not state the fixative (caption: fixative unreported). The paraffin-section example used heat retrieval in EDTA at pH 8.0, but it does not establish that retrieval is required under every condition (caption: EDTA retrieval). For IF/ICC, HPA reports nucleoplasmic XRCC4 signal, but the supplied evidence does not show whether IF or frozen sections are easier than paraffin IHC; lipid loss during paraffin processing can leave adipocytes with thin cytoplasmic rims that complicate scoring (HPA: Nucleoplasm enhanced; standard histology practice).
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 by checking retrieval, nuclear localisation, controls and scoring before interpreting biological differences.
How should I adjust retrieval when XRCC4 nuclear staining is weak?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A00787-2). The catalog antibody produced tissue staining after that retrieval, followed by 2 μg/ml primary antibody overnight at 4°C; those conditions provide a reference when investigating weak signal (datasheet A00787-2). Check whether section thickness, heating and cooling were consistent across slides, then compare a known staining control on the same run (standard IHC practice). If staining remains weak, test a different retrieval condition on matched sections and judge both nuclear signal and background; the alternative is a troubleshooting experiment, not a documented XRCC4 requirement (standard IHC practice).
Could fixation explain variable XRCC4 staining between paraffin blocks?
The selected paraffin-section caption does not report a fixative, so XRCC4-specific sensitivity to fixation is unknown for this antibody (datasheet A00787-2). Record each block’s fixative and processing history, then compare sections processed and stained together before assigning differences to biology (standard IHC practice). If archival blocks vary, include a consistent reference section in each run and inspect nuclear morphology for evidence of uneven processing (standard IHC practice). Keep the documented EDTA retrieval at pH 8.0 and 2 μg/ml primary concentration constant during that comparison, changing one preparation variable at a time (datasheet A00787-2; standard IHC practice).
What XRCC4 staining pattern should I expect in tissue sections?
Expect predominantly nuclear staining: tissue IHC reports ubiquitous nuclear expression, and cell imaging places XRCC4 in the nucleoplasm (HPA tissue IHC; HPA subcellular). UniProt also annotates chromosome and cytoplasm localisation and recruitment to double-strand break sites, so compartment alone cannot establish whether an unusual pattern is specific (UniProt Q13426). Evaluate nuclear signal within intact, identifiable cells against a reference tissue and the local background on the same slide (standard IHC practice). If staining is mainly diffuse cytoplasmic or extracellular, review retrieval, chromogen development and morphology before treating it as XRCC4 redistribution (HPA tissue IHC; standard IHC practice).
Could isoforms or modifications change the IHC signal?
XRCC4 has 3 annotated isoforms, and its record lists phosphorylated residues including Ser-53, Ser-193 and Thr-233 (UniProt Q13426). The supplied catalog caption does not identify the antibody epitope or establish whether it recognises every isoform or modification state (datasheet A00787-2). Treat differences in nuclear intensity as staining differences until epitope information or an independent specificity experiment supports a molecular explanation (standard IHC practice). When comparing samples, hold the documented EDTA pH 8.0 retrieval and 2 μg/ml primary concentration constant so processing variation does not obscure the question (datasheet A00787-2; standard IHC practice).
How can IF help investigate an ambiguous IHC pattern?
Use IF/ICC as a separate localisation check: cell imaging places XRCC4 in the nucleoplasm, while the paraffin-section caption documents a chromogenic IHC workflow (HPA subcellular; datasheet A00787-2). For tissue multiplexing, pair XRCC4 with a validated marker for the cell population being assessed, such as respiratory epithelial cells when examining bronchus, and check each channel separately (HPA tissue IHC; standard IF practice). Choose a fluorophore away from the tissue’s strongest autofluorescence, using a far-red channel if its background is lower (standard IF practice). Because XRCC4 is intracellular and has no transmembrane segment, optimise permeabilisation to permit nuclear antibody access, with fixation effects assessed directly in the IF preparation (UniProt Q13426; standard IF practice).
How do I reduce diffuse brown background without losing nuclear signal?
First distinguish diffuse deposit from staining confined to intact nuclei, the tissue pattern reported for XRCC4 (HPA tissue IHC). The catalog example used a 10% goat serum block, 2 μg/ml primary overnight at 4°C, a peroxidase-conjugated secondary for 30 minutes at 37°C, and DAB development (datasheet A00787-2). Compare a primary-omission control and a consistently staining reference section while checking washes, secondary binding and DAB development (standard IHC practice). Apply an endogenous peroxidase block as part of the chromogenic workflow and shorten excessive development if control sections show deposit; neither step demonstrates XRCC4 specificity by itself (standard IHC practice).
How should I score XRCC4 when many cell types stain? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and nuclear compartment before scoring, because tissue IHC reports broad nuclear XRCC4 expression with differences among cell types (HPA tissue IHC). For each region, record the percentage of positive nuclei and an intensity-based H-score, keeping staining thresholds and image settings consistent within the study (standard IHC practice). Normalise counts to the number of evaluable nuclei in that cell population; if reporting positive-cell density, use the assessed tissue area in mm² and exclude non-evaluable regions (standard IHC practice). Include a reference section across staining runs, and report how background and counterstain affected the threshold so apparent shifts can be assessed (standard IHC practice).
When is an apparent XRCC4 positive likely to be artefactual?
A plausible positive is staining in intact nuclei: HPA reports ubiquitous nuclear expression, with high staining in bronchial respiratory epithelial cells and no detection in liver cholangiocytes (HPA tissue IHC). Use those cell-level observations as context rather than assuming every cell in either tissue must match the reported category (HPA tissue IHC; standard IHC practice). Be cautious with signal restricted to section edges, necrotic areas or extracellular material, or with a chiefly cytoplasmic pattern that conflicts with the reported nuclear profile (HPA tissue IHC; standard IHC practice). Check a primary-omission control for endogenous enzyme or detection background and compare intact cells before calling an unusual signal XRCC4 (standard IHC practice).
Boster reagents
Best XRCC4 / DNA repair protein XRCC4 IHC Antibodies
The human-reactive XRCC4 catalog antibodies have IHC images from paraffin-embedded cancer sections (catalog IHC captions); A00787-2 also has IF images from U20S cells and a paraffin-embedded tissue section (A00787-2 IF captions).
Real IHC data
Anti-XRCC4 Antibody ®
Cat # A00787-2
Real IHC data
Anti-XRCC4 Antibody ®
Cat # PB9908
A00787-2 shows IHC in human lung squamous cell, breast duct, colorectal and liver cancer sections, plus IF in U20S cells and a human colorectal adenocarcinoma section (A00787-2 image captions). PB9908 shows IHC in a human intestinal cancer section (PB9908 IHC caption).
Which to pick: For paraffin-section IHC, both have chromogenic examples: A00787-2 uses 2 μg/ml with EDTA pH 8.0 retrieval and DAB in human lung squamous cell carcinoma (A00787-2 IHC caption); PB9908 uses 1 μg/ml with the same retrieval buffer and chromogen in human intestinal cancer (PB9908 IHC caption). For IF/ICC, choose A00787-2: both applications are listed and IF images are provided (A00787-2 applications and IF captions); PB9908 lists neither application (PB9908 applications). Neither has documented cross-species reactivity here because both list Human only (catalog reactivity); clonality is unreported (catalog entries), as is the fixative used for either pictured IHC section (IHC captions).
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 staining in tissue IHC (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: Cholangiocytes may lack detectable staining (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope coverage is unspecified (UniProt); Regulation: Widely expressed (UniProt). Evidence: UniProt Q13426, HPA tissue IHC · Supported, HPA ICC-IF · nucleoplasm., Datasheet A00787-2, PMC5675685, PMC8425215. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/XRCC4/immunohistochemistry-ihc-guide.