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ERCC5 / DNA excision repair protein ERCC-5 · IHC design guide
Design Immunohistochemistry for ERCC5
Plan ERCC5 chromogenic IHC in paraffin sections using the nuclear and cytoplasmic tissue pattern (HPA tissue IHC). Compare high staining in colon glandular cells with undetected staining in hippocampal glial cells (HPA tissue IHC), starting the catalog antibody at 2–5 μg/ml (datasheet A01770-2).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ERCC5 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 and cytoplasmic tissue staining (HPA tissue IHC) | |
| Staining pattern | Widespread nuclear and cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A01770-2) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Hippocampus |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A01770-2) | |
| Caveat | HPA staining may reflect proteins from multiple genes (HPA tissue IHC) | |
| Regulation | Low tissue specificity (HPA tissue RNA) | |
| Isoform / epitope | 3 isoforms; epitope coverage unknown (UniProt) |
Section 1
Recommended ERCC5 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is paired with one published ERCC5 IHC protocol for paraffin sections of human or rat intervertebral discs (PMC12261858).
| Sample | Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A01770-2) |
| Fixation | Image fixative and duration unreported (datasheet A01770-2); 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 A01770-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A01770-2) |
| Primary antibody | Rabbit anti-ERCC5, 2-5 μg/ml (datasheet A01770-2) |
| Primary incubation | Overnight at 4 °C (datasheet A01770-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A01770-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ERCC5-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear and cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet: A01770-2); the published protocol reports antigen retrieval without specifying conditions (PMC12261858).
Section 2
What Is the Expected ERCC5 Staining Pattern?
ERCC5 is a nuclear DNA repair protein without a transmembrane segment (UniProt P28715: location and topology). In tissue IHC, expect staining in many cell types, including strong staining of appendix, colon, and duodenal glandular cells (HPA tissue IHC: High). HPA also reports widespread cytoplasmic staining, but rates tissue results Supported with medium consistency and cautions that the antibodies target proteins from more than one gene (HPA tissue IHC: profile and reliability).
| Glandular cells show a clear nuclear component with strong overall staining in appendix, colon, or duodenum. | This fits ERCC5's nuclear location and the reported high staining in these glandular cells (UniProt P28715: location; HPA tissue IHC: High). Score the nuclear component separately from cytoplasmic color so the result remains interpretable despite HPA's broader nuclear and cytoplasmic profile (HPA tissue IHC: profile). |
| Color is confined to cell borders, luminal material, or extracellular areas, with no convincing nuclei. | A border-only or extracellular pattern does not fit a nuclear protein without a transmembrane segment (UniProt P28715: location and topology). Treat it as a potential staining artefact and review morphology and controls (general IHC practice). Cytoplasmic color alone is less decisive because HPA reports it in tissue IHC (HPA tissue IHC: profile). |
| Hippocampal glial cells stain strongly, especially when the expected glandular cells are weak. | HPA reports hippocampal glial cells as not detected, while several glandular populations stain High (HPA tissue IHC: cell-level results). Investigate antibody cross-reactivity or endogenous chromogenic activity with appropriate controls (general IHC practice). Do not generalize this finding to all glia: caudate glial cells are reported Low (HPA tissue IHC: cell-level results). |
| Brown color spreads across nuclei, cytoplasm, and tissue spaces without a clear cell-level pattern. | Diffuse color cannot establish ERCC5 localization, even though HPA describes widespread nuclear and cytoplasmic expression (HPA tissue IHC: profile). Compare a no-primary control and inspect wash and blocking performance before scoring specific staining (general IHC practice); HPA's multi-gene antibody caution increases the need for restraint (HPA tissue IHC: reliability). |
| A high-staining glandular tissue has no convincing cellular signal. | An absent result in appendix, colon, or duodenal glandular cells conflicts with the reported High IHC staining (HPA tissue IHC: cell-level results). First check tissue preservation, retrieval, antibody and detection controls, and counterstain visibility (general IHC practice). This result alone cannot identify the failed step or prove ERCC5 is absent. |
💡Expected ERCC5 appearanceCall the result consistent with ERCC5 when high-staining glandular cells show clear cellular staining with an interpretable nuclear component (HPA tissue IHC: High; UniProt P28715: location); border-only or extracellular color without nuclei is suspect (UniProt P28715: location and topology; general IHC practice), while cytoplasmic color needs cautious interpretation because HPA also reports it (HPA tissue IHC: profile and reliability).
| Choice of tissue and cell population | Appendix, colon, duodenum, gallbladder, rectum, and small intestine have High staining in glandular cells (HPA tissue IHC: cell-level results). Hippocampal glia are reported not detected, while caudate glia are Low (HPA tissue IHC: cell-level results). Evaluate the named cells within each section; a tissue label alone is insufficient. |
| Compartment and specificity | UniProt places ERCC5 in the nucleus and chromosome, whereas HPA tissue IHC describes ubiquitous nuclear and cytoplasmic staining (UniProt P28715: location; HPA tissue IHC: profile). HPA rates the tissue evidence Supported, notes medium RNA agreement, and cautions that antibodies target proteins from more than one gene (HPA tissue IHC: reliability). Cytoplasmic signal therefore warrants cautious scoring. |
| Antibody validation | HPA045845 and HPA050374 are each rated Supported for IHC (HPA antibodies: IHC validation). That rating supports using the observed tissue pattern as a comparison, while the tissue-level multi-gene caution still applies (HPA tissue IHC: reliability). It does not establish that every positive cell or compartment contains only ERCC5. |
| Isoforms and processing | UniProt lists three ERCC5 isoforms, one annotated chain spanning residues 1–1186, and no signal peptide or propeptide (UniProt P28715: isoforms and processing). The supplied record gives no antibody epitope, so isoform coverage cannot be predicted. Its lack of a transmembrane segment offers no basis to expect a cell-surface staining pattern (UniProt P28715: topology). |
| IF/ICC Q&A: where should signal appear? | In IF/ICC, look for nucleoplasmic signal; HPA lists the nucleoplasm as the supported main location and shows images for U-251MG, A-431, and U2OS (HPA subcellular ICC-IF). This observation carries HPA's caution about antibodies targeting proteins from multiple genes (HPA subcellular ICC-IF). IF/ICC conditions belong to its separate guide. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected glandular cells are unstained. | A run or specimen problem is possible; HPA reports High staining in several glandular populations (HPA tissue IHC: cell-level results). | Check section morphology and the run's positive, negative, and detection controls; review the established retrieval and antibody conditions (general IHC practice). Do not infer target-specific fixation sensitivity: none is supplied. |
| Only cytoplasm stains. | HPA reports cytoplasmic expression, but UniProt places ERCC5 in the nucleus; HPA also cautions about multi-gene antibody targeting (HPA tissue IHC: profile and reliability; UniProt P28715: location). | Record cytoplasmic and nuclear scores separately, compare controls and morphology, and avoid calling the cytoplasmic component uniquely ERCC5 on this evidence (general IHC practice; HPA tissue IHC: reliability). |
| Hippocampal glial cells show strong color. | That differs from HPA's not-detected result for those cells; nonspecific binding or endogenous detection activity are possible explanations (HPA tissue IHC: cell-level results; general IHC practice). | Examine a no-primary control and the relevant blocking controls, then compare the same run with a reported high-staining glandular tissue (general IHC practice; HPA tissue IHC: cell-level results). |
| Brown deposit follows edges or tissue spaces. | That distribution conflicts with UniProt's nuclear location and lack of a transmembrane segment; deposit or background is possible (UniProt P28715: location and topology; general IHC practice). | Inspect section integrity, reagent coverage, washes, and no-primary control before assigning a cellular score (general IHC practice). |
| Most cells are uniformly dark and nuclei cannot be distinguished. | Overdeveloped chromogen or high background can obscure compartment assessment (general IHC practice); HPA's broad tissue profile alone cannot resolve specificity (HPA tissue IHC: profile and reliability). | Review chromogen development, blocking, and the no-primary control; score only cells whose boundaries and nuclear compartment remain interpretable (general IHC practice). |
| Two IHC antibodies give different cell patterns. | Both listed antibodies are IHC Supported, while HPA warns of multi-gene targeting and only medium consistency with RNA data (HPA antibodies: IHC validation; HPA tissue IHC: reliability). | Compare their nuclear and cytoplasmic patterns in the same named cell populations and controls; report discordance rather than merging the results into one ERCC5-specific score (general IHC practice; HPA tissue IHC: reliability). |
Experiment evidence
IHC & IF images showing ERCC5 staining
A01770-2 · IHCIHC analysis of ERCC5 using anti-ERCC5 antibody (A01770-2). ERCC5 was detected in a paraffin-embedded section of human colon cancer tissue.
A01770-2 · IHCIHC analysis of ERCC5 using anti-ERCC5 antibody (A01770-2). ERCC5 was detected in a paraffin-embedded section of human colon tissue. Heat me
A01770-2 · IFIF analysis of ERCC5 using anti-ERCC5 antibody (A01770-2) and anti-Beta Tubulin antibody (M01857-3). ERCC5 was detected in an immunocytochem
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000134899-ERCC5/tissue/cerebral+cortex#imid_12414373
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000134899-ERCC5/tissue/colon#imid_12414291
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000134899-ERCC5/tissue/liver#imid_12414300
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000134899-ERCC5/tissue/kidney#imid_12414293
HPA · ICC-IF U-251MGHuman Protein Atlas immunofluorescence, U-251MG, antibody HPA045845; green = ERCC5, blue = nucleus, red = microtubules.
HPA · ICC-IF U-251MGHuman Protein Atlas immunofluorescence, U-251MG, antibody HPA045845; green = ERCC5, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ERCC5 IHC & IF
🧪Run colon first and assess glandular cells for ERCC5 staining (HPA: High in colon glandular cells; selected-SKU caption: colon tissue IHC). Use hippocampal glial cells as the biological comparison (HPA: Not detected in hippocampal glial cells); on the colon slide, cells judged negative should show counterstain without specific nuclear DAB, but do not presume that all stromal cells are negative (standard IHC practice).
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Hippocampus (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ERCC5 in U-251MG, A-431, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and species- and format-matched rabbit IgG isotype controls, plus an ERCC5-knockout specimen if available (selected-SKU caption: rabbit primary antibody; standard IHC practice). Quench endogenous peroxidase and check for nonspecific DAB signal in colon before scoring (selected-SKU caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU caption: fixative not stated). That caption uses heat retrieval in EDTA at pH 8.0, which supports this starting condition but does not establish that ERCC5 detection requires it (selected-SKU caption: EDTA pH 8.0 retrieval). IHC-P has a supplied colon-section procedure; the evidence does not establish that frozen sections or IF/ICC are easier, and endogenous peroxidase background warrants attention with HRP/DAB in colon (selected-SKU caption: colon-section HRP/DAB procedure; standard IHC practice).
HPA tissue IHC evidence for ERCC5
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — Medium consistency between antibody staining and RNA expression data. Caution, targets protein from more than one gene.). 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 |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Fallopian tube | Ciliated cells (cell body) | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Hippocampus | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ERCC5 IHC Tips
Use the catalog antibody’s paraffin-section result as the starting point, then judge ERCC5 staining by compartment, tissue context, and controls (datasheet A01770-2; UniProt P28715; HPA tissue IHC).
Which retrieval conditions should I try first for ERCC5 in paraffin sections?
Begin with heat-mediated antigen retrieval in EDTA at pH 8.0 for chromogenic ERCC5 IHC (datasheet A01770-2). The selected paraffin-section result used that retrieval before 10% goat-serum blocking and 2 μg/ml primary antibody overnight at 4°C (datasheet A01770-2). If staining is weak, compare a longer and shorter heating interval on adjacent sections while keeping detection and exposure conditions matched (standard IHC practice). Record the retrieval interval and inspect tissue integrity alongside nuclear signal, since excessive heating can damage sections or raise background (standard IHC practice; UniProt P28715 localisation).
How should I troubleshoot weak ERCC5 staining when fixation history is uncertain?
The selected ERCC5 image documents a paraffin section but does not state its fixative, so target-specific fixation sensitivity is unknown (datasheet A01770-2). Record the fixative and fixation duration for each specimen, then compare similarly processed sections before attributing a weak result to ERCC5 abundance (standard IHC practice). Run the documented EDTA pH 8.0 retrieval and 2 μg/ml antibody condition on a reference section processed alongside the test tissue (datasheet A01770-2; standard IHC practice). If fixation histories differ, compare staining within matched processing groups and flag any group difference as a technical possibility (standard IHC practice).
Should ERCC5 staining appear in nuclei, cytoplasm, or discrete foci?
Prioritise nuclear staining when assessing ERCC5 because UniProt places it in the nucleus and on chromosomes, and HPA reports supported nucleoplasmic localisation (UniProt P28715 localisation; HPA subcellular). ERCC5 can colocalise with RAD51 and BRCA2 in nuclear foci during replication stress, but those observations do not make foci obligatory in routine tissue sections (UniProt P28715, PubMed:26833090). HPA tissue IHC also describes a ubiquitous nuclear and cytoplasmic pattern, with a caution that its antibody targets proteins from more than one gene (HPA tissue IHC). Assess staining against morphology and a control section before treating diffuse cytoplasmic DAB as specific ERCC5 (standard IHC practice; HPA tissue IHC).
Could isoforms or epitope accessibility explain discordant ERCC5 staining?
ERCC5 has 3 annotated isoforms and an intact protein spanning residues 1–1186, so epitope coverage matters when comparing antibodies (UniProt P28715 isoforms and processing). The supplied catalog image does not identify the antibody epitope, preventing an isoform-specific interpretation of its DAB pattern (datasheet A01770-2). Phosphoserines at residues 384 and 705 are annotated, but the payload does not show that either changes IHC recognition (UniProt P28715 modified residues; datasheet A01770-2). If antibodies disagree, check their disclosed immunogen regions and compare compartment-specific staining under matched retrieval and detection conditions (standard IHC practice; UniProt P28715 localisation).
How can I investigate ERCC5 localisation with multiplex IF?
For a separate IF assay, pair ERCC5 with a marker identifying the cell population of interest and a nuclear counterstain so foci can be assigned to individual nuclei (standard IF practice; UniProt P28715 localisation). HPA reports supported nucleoplasmic localisation, while UniProt describes RAD51 and BRCA2 colocalisation in nuclear foci under replication stress (HPA subcellular; UniProt P28715, PubMed:26833090). Choose spectrally separated fluorophores after checking tissue autofluorescence and include single-stain controls to detect channel bleed-through (standard IF practice). Because ERCC5 has no transmembrane segment and its expected signal is intracellular, permeabilise cells for access to the nuclear epitope, then verify localisation with controls (UniProt P28715 topology and localisation; standard IF practice).
What should I adjust when ERCC5 DAB staining is diffuse or widespread?
First inspect a no-primary control and the distribution of DAB, including section edges and damaged areas, before changing the antibody concentration (standard IHC practice). The selected paraffin-section result used 10% goat serum, 2 μg/ml primary antibody overnight at 4°C, and a peroxidase-conjugated secondary for 30 minutes at 37°C (datasheet A01770-2). Include a peroxidase-blocking step and assess DAB development time as general chromogenic IHC controls (standard IHC practice). Widespread cytoplasmic signal requires particular scrutiny because HPA reports both nuclear and cytoplasmic staining but warns of cross-gene antibody targeting (HPA tissue IHC).
How should I score ERCC5 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored compartment before analysis, using nuclear staining as the primary readout and recording cytoplasmic staining separately when present (UniProt P28715 localisation; HPA tissue IHC). An H-score can combine the percentage of cells at each intensity level from 0–3, while percentage-positive cells offers a simpler measure (standard IHC practice). Normalise each score to the number of evaluable cells in the same annotated compartment, and exclude folds, necrosis, and section edges using consistent rules (standard IHC practice). Keep retrieval, DAB development, imaging, and scoring thresholds matched across specimens; report the number of evaluable cells and replicate sections (standard IHC practice).
How can I distinguish true ERCC5 positivity from staining artefact?
A credible result has interpretable cellular morphology and nuclear signal consistent with ERCC5 localisation, while any cytoplasmic component needs separate assessment (UniProt P28715 localisation; HPA tissue IHC). Colon glandular cells are reported as high by HPA, and the selected catalog image shows ERCC5 detection in a paraffin section of human colon cancer (HPA tissue IHC; datasheet A01770-2). Compare suspected signal with no-primary and peroxidase controls, especially where DAB follows section edges, necrosis, or endogenous enzyme activity (standard IHC practice). Interpret tissue-wide claims cautiously because HPA rates its tissue IHC evidence Supported but warns that its antibody targets proteins from more than one gene (HPA tissue IHC).
Boster reagents
Best ERCC5 / DNA excision repair protein ERCC-5 IHC Antibodies
The IHC-validated anti-ERCC5 antibody has images from human paraffin-embedded colon and colon cancer sections, plus IF images from HeLa cells and a human colon cancer section (catalog image captions).
Real IHC data
Anti-XPG/ERCC5 Antibody ®
Cat # A01770-2
A01770-2 has IHC images from paraffin-embedded human colon and colon cancer sections (catalog IHC captions). A01770-2 also has IF images from HeLa cells and a paraffin-embedded human colon cancer section (catalog IF captions).
Which to pick: Choose A01770-2 for paraffin-section IHC; its caption documents EDTA retrieval at pH 8.0 and a primary concentration of 2 μg/ml (A01770-2 IHC caption). For IF/ICC, the same SKU lists both applications and an IF concentration of 5 μg/ml (catalog applications and dilution); its clone status is unreported (catalog clone field). The catalog lists Human, Mouse and Rat reactivity (catalog reactivity), but the supplied IHC captions show human sections only, and their fixative is unreported (A01770-2 IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P28715 (ERCC5_HUMAN, DNA excision repair protein ERCC-5).
- Human Protein Atlas. ERCC5 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. ERCC5 subcellular location (ICC-IF): Localized to the nucleoplasm. Caution: Based on antibodies targeting proteins from multiple genes..
- Human Protein Atlas. ERCC5 antibody validation summary (2 antibodies).
- Targeted Inhibition of cGAS/STING signaling induced by aberrant R-Loops in the nucleus pulposus to alleviate cellular senescence and intervertebral disc degeneration. Journal of nanobiotechnology 2025 — PMC12261858.
- Association between nucleotide excision repair gene polymorphism and colorectal cancer risk. Journal of clinical laboratory analysis 2019 — PMC6805325.
- Identification of a Splenic Marginal Zone Lymphoma Signature: Preliminary Findings With Diagnostic Potential. Frontiers in oncology 2020 — PMC7225304.
- PubMed PMID:8483504 — UniProt-cited evidence.
- PubMed PMID:7510366 — UniProt-cited evidence.
- PubMed PMID:8413238 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ERCC5 (DNA excision repair protein ERCC-5). Expected localisation Nuclear and cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A01770-2). Validated IHC antibody: A01770-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Hippocampus. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A01770-2); 2. Fixation: Image fixative and duration unreported (datasheet A01770-2); 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 A01770-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A01770-2); 8. Primary antibody: Rabbit anti-ERCC5, 2-5 μg/ml (datasheet A01770-2); 9. Primary incubation: Overnight at 4 °C (datasheet A01770-2). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A01770-2); Caveat: HPA staining may reflect proteins from multiple genes (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope coverage unknown (UniProt); Regulation: Low tissue specificity (HPA tissue RNA). Evidence: UniProt P28715, HPA tissue IHC · Supported, HPA ICC-IF · nucleoplasm. Caution: Based on antibodies targeting proteins from…, Datasheet A01770-2, PMC12261858. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ERCC5/immunohistochemistry-ihc-guide.