This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
RPA1 / Replication protein A 70 kDa DNA-binding subunit · IHC design guide
Design Immunohistochemistry for RPA1
Plan paraffin-section RPA1 IHC around general nuclear staining (HPA tissue IHC). The catalog antibody M01317-1 provides a 1:50 starting dilution for chromogenic IHC (datasheet M01317-1).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RPA1 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 | General nuclear staining in tissue (HPA tissue IHC) | |
| Staining pattern | Nuclear staining across diverse tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M01317-1) | |
| Positive control | Adipose tissue+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | DNA damage may produce nuclear repair foci (UniProt) | |
| Regulation | No tissue-specific regulation annotated (UniProt) | |
| Isoform / epitope | No isoforms annotated; two chains differ at the N terminus (UniProt) |
Section 1
Recommended RPA1 IHC & IF Protocols
The catalog antibody uses heat-mediated EDTA pH 8.0 retrieval (datasheet: M01317-1). Four published RPA1 IHC protocols provide tissue-specific examples (PMC10063624; PMC12235235; PMC12925576; PMC6139602).
| Sample | Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet M01317-1) |
| Fixation | Image fixative and duration unreported (datasheet M01317-1); 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 M01317-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M01317-1) |
| Primary antibody | Rabbit monoclonal (clone BB-18) anti-RPA1, 1:50 (datasheet M01317-1) |
| Primary incubation | Overnight at 4 °C (datasheet M01317-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet M01317-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | RPA1-positive staining in adipocytes of adipose tissue (HPA tissue IHC: High). HPA tissue profile: General nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 for the catalog antibody (datasheet: M01317-1). The published protocols used citrate pH 6.0 with their respective antibodies (PMC10063624; PMC12235235; PMC12925576; PMC6139602).
Section 2
What Is the Expected RPA1 Staining Pattern?
RPA1 should stain nuclei across multiple cell types in paraffin sections (HPA tissue IHC: general nuclear expression). High staining is reported in adipocytes, glandular cells, hematopoietic cells, respiratory epithelial cells, glial cells and neurons (HPA tissue IHC). The tissue pattern is Supported, with medium consistency between staining and RNA data (HPA tissue IHC). RPA1 has no transmembrane segment (UniProt P27694 topology).
| Distinct nuclear staining in the documented cell types, including breast glandular and bone marrow hematopoietic cells (HPA tissue IHC). | This matches the reported general nuclear pattern and High staining in those cells (HPA tissue IHC). Interpret intensity alongside the section controls; the HPA tissue assessment has medium consistency with RNA data (HPA tissue IHC). |
| Predominantly cytoplasmic or membrane staining, with little nuclear signal. | This conflicts with the nuclear tissue pattern (HPA tissue IHC) and nuclear localization (UniProt P27694). Check whether the apparent signal follows tissue structures or the detection reaction, then reassess antibody specificity with controls (general IHC practice). |
| Strong staining is confined to an unexpected cell population while documented positive cells are unstained. | The mismatch raises concern for cross-reactivity or endogenous chromogenic activity (general IHC practice). HPA reports Low tissue specificity and lists no negative tissue in this payload, so an unexpected cell alone cannot establish a false positive (HPA tissue IHC). |
| Diffuse brown color obscures nuclei or extends across tissue and blank areas. | This is background rather than a readable nuclear result (general IHC practice). Check blocking, washes and the detection-only control before assigning cell-level RPA1 staining (general IHC practice). |
| No nuclear signal in a documented High-staining cell population (HPA tissue IHC). | Treat the run as unresolved until a positive control and detection controls perform as expected (general IHC practice). The HPA level describes observed staining; it does not establish that every section or antibody condition must give the same intensity (HPA tissue IHC). |
💡Expected RPA1 appearanceA convincing positive is predominantly nuclear, with High staining in documented cell populations (HPA tissue IHC); dominant cytoplasmic or membrane color is discordant with RPA1 localization (UniProt P27694; HPA tissue IHC).
| Tissue and cell selection | HPA reports High staining in adipocytes; adrenal and breast glandular cells; marrow hematopoietic cells; bronchial respiratory epithelial cells; caudate glia; cerebellar Purkinje cells; and cortical neurons (HPA tissue IHC). These are candidate positive controls, with no negative tissue supplied (HPA tissue IHC). |
| Evidence strength | The tissue pattern is Supported with medium staining–RNA consistency, and RNA has Low tissue specificity (HPA tissue IHC). Assess compartment and controls together rather than treating staining intensity alone as proof of identity (general IHC practice). |
| Antibody validation | HPA006914 and CAB004563 have Supported IHC status (HPA antibodies). HPA046497 has Enhanced ICC status but no IHC status in the supplied list (HPA antibodies); validation for one application does not establish performance in another (general IHC practice). |
| Topology and protein forms | RPA1 lacks a transmembrane segment, signal peptide and propeptide; UniProt lists full-length and N-terminally processed chains, with no annotated isoforms (UniProt P27694). These annotations support a non-membrane expectation but do not identify the antibody epitope or predict a staining difference (UniProt P27694). |
| IF/ICC: where should signal appear? | The expected location is enhanced nucleoplasmic staining (HPA subcellular ICC-IF). UniProt also records nuclear PML-body enrichment in cells displaying alternative lengthening of telomeres (UniProt P27694); that conditional pattern should not be required in routine tissue IHC. |
| Antigen retrieval | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. |
| Situation | Likely cause | Next action |
|---|---|---|
| No staining in a documented High-staining population (HPA tissue IHC). | A failed primary, retrieval or detection step is possible (general IHC practice); the supplied sources do not identify an RPA1-specific fixation effect. | Check the positive control and each detection step, then review the selected antibody's IHC conditions before changing retrieval (general IHC practice). |
| Cytoplasmic or membrane color dominates. | The compartment disagrees with nuclear RPA1 (UniProt P27694; HPA tissue IHC); background or off-target binding is possible (general IHC practice). | Compare with a detection-only control and assess nuclear counterstain alignment; repeat with an IHC-supported antibody if the mismatch persists (general IHC practice; HPA antibodies). |
| Brown color appears broadly across tissue or outside cells. | Nonspecific binding or chromogenic background may obscure nuclear staining (general IHC practice). | Review blocking, washes and detection-only controls; score RPA1 only where cell nuclei remain distinguishable (general IHC practice). |
| Only unexpected cells stain while documented positive cells do not. | Cross-reactivity or endogenous detection activity is possible (general IHC practice); the HPA payload provides no negative tissue comparator (HPA tissue IHC). | Check tissue identity, cell morphology and detection-only controls; compare against a documented High-staining cell population before interpreting specificity (general IHC practice; HPA tissue IHC). |
| Nuclear intensity varies across one section. | Section or staining variability may contribute (general IHC practice); the supplied sources do not establish an RPA1-specific fixation explanation. | Compare adjacent regions and the positive control, and record the distribution rather than assigning a biological cause from intensity alone (general IHC practice). |
| Punctate nuclear staining is absent despite a clear nuclear signal. | PML-body enrichment is conditional on cells displaying alternative lengthening of telomeres (UniProt P27694). | Evaluate the general nuclear tissue pattern first (HPA tissue IHC); do not require PML-body puncta for a positive paraffin-section result (UniProt P27694). |
Experiment evidence
IHC & IF images showing RPA1 staining
M01317-1 · IHCIHC analysis of RPA1 using anti-RPA1 antibody (M01317-1). RPA1 was detected in a paraffin-embedded section of human breast cancer tissue. He
M01317-1 · IHCIHC analysis of RPA1 using anti-RPA1 antibody (M01317-1). RPA1 was detected in a paraffin-embedded section of human colon cancer tissue. Hea
M01317-2 · IHCIHC analysis of RPA70 using anti-RPA70 antibody (M01317-2). RPA70 was detected in paraffin-embedded section of human intestinal cancer tissu
M01317-2 · IHCIHC analysis of RPA70 using anti-RPA70 antibody (M01317-2). RPA70 was detected in paraffin-embedded section of human lung cancer tissue. Hea
M01317-2 · IFIF analysis of RPA70 using anti-RPA70 antibody (M01317-2). RPA70 was detected in immunocytochemical section of A549 cells. Enzyme antigen re
PB9886 · IFIF analysis of RPA70 using anti-RPA70 antibody (PB9886). RPA70 was detected in an immunocytochemical section of A549 cells. Enzyme antigen r
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000132383-RPA1/tissue/cerebral+cortex#imid_2156824
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000132383-RPA1/tissue/colon#imid_2156742
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000132383-RPA1/tissue/liver#imid_2156743
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000132383-RPA1/tissue/kidney#imid_2156736
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA046497; green = RPA1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA046497; green = RPA1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RPA1 IHC & IF
🧪Run breast tissue first and assess nuclear staining in glandular cells (HPA: High in breast glandular cells; UniProt P27694: nucleus). HPA detects RPA1 in all 45 scored tissues and lists no negative tissue, so no-primary and isotype controls carry the negative comparison; any anucleate erythrocytes present should lack nuclear staining, while unstained stromal nuclei should not be assumed to be RPA1-negative (HPA: no negative tissue; standard IHC practice).
Positive control tissue: Adipose tissue (Adipocytes, HPA High)
Negative control tissue: None in HPA: RPA1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RPA1 in A-431, A-549, U2OS, HeLa BAC 4595, HeLa , with annotated localisation: Nucleoplasm (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide, a matched rabbit IgG isotype control, and, if available, an RPA1 knockout specimen or cognate peptide block (caption: rabbit primary and anti-rabbit secondary; standard IHC practice). Quench endogenous peroxidase before HRP/DAB detection, particularly where blood is present in the breast section (caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected M01317-1 tissue-IHC caption does not state the fixative (caption: fixative unreported). The caption uses heat retrieval in EDTA at pH 8.0 and a 1:50 primary dilution overnight at 4°C, but does not establish whether retrieval is required (caption: M01317-1 breast cancer IHC). HPA reports nucleoplasmic ICC-IF staining, but the supplied evidence does not establish whether frozen sections or IF are easier than paraffin IHC; blood in a breast section may add peroxidase-related DAB background (HPA: nucleoplasm enhanced; standard IHC practice).
HPA tissue IHC evidence for RPA1
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 → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: RPA1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced RPA1 IHC Tips
Use nuclear staining, tissue controls, and the selected antibody’s paraffin-section conditions to troubleshoot RPA1 chromogenic IHC (HPA tissue IHC; caption M01317-1).
How should I adjust retrieval when RPA1 nuclear staining is weak?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet M01317-1). The selected tissue-IHC image used this retrieval before an overnight 1:50 primary incubation at 4°C (caption M01317-1). If staining is weak, vary heating duration on adjacent sections while keeping antibody dilution and detection constant (general IHC practice). Check nuclear detail and tissue adhesion after each trial, because damaged morphology makes nuclear scoring unreliable (general IHC practice). Judge improvement by clearer nuclear staining, the expected pattern for RPA1 (HPA tissue IHC: general nuclear expression).
What fixation conditions should I use if RPA1 staining varies between specimens?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative (caption M01317-1). Record fixative, concentration, and time for each specimen, then compare matched sections using the same EDTA pH 8.0 retrieval and detection conditions (general IHC practice; datasheet M01317-1). Keep processing and section thickness consistent, and assess nuclear preservation before comparing signal intensity (general IHC practice). Nuclear staining is the expected readout (UniProt P27694: nucleus; HPA tissue IHC: general nuclear expression). Neither RPA1’s modifications nor its lack of a transmembrane segment establishes this antibody’s fixation sensitivity (UniProt P27694: modified residues and topology).
Should diffuse cytoplasmic or punctate RPA1 staining count as positive?
Score convincing nuclear staining first, since tissue IHC shows general nuclear expression and subcellular imaging places RPA1 in the nucleoplasm (HPA tissue IHC; HPA subcellular: nucleoplasm). Diffuse cytoplasmic DAB without corresponding nuclear signal warrants a review of background, counterstain, and the no-primary control (general IHC practice). Nuclear puncta can be plausible, but UniProt specifically reports PML-body enrichment in cells displaying alternative lengthening of telomeres (UniProt P27694: subcellular location). Do not infer that mechanism from puncta alone; document their frequency and confirm their identity with an appropriate colocalisation experiment (general IF practice).
Could epitope changes explain inconsistent RPA1 staining across sections?
The supplied record lists 0 isoforms and two annotated chains beginning at residues 1 and 2 (UniProt P27694: isoforms and processing). It also lists modifications including acetylation near residues 163 and 167 and phosphorylation at 180 and 191 (UniProt P27694: modified residues). The antibody’s binding epitope is unspecified, so these annotations cannot establish an epitope-specific cause of staining differences (record: epitope unspecified). Compare serial sections with matched retrieval and antibody conditions, and seek documented epitope information before attributing a loss of signal to processing or modification (general IHC practice).
How can IF help verify an uncertain RPA1 IHC pattern?
For a separate IF/ICC cross-check, multiplex RPA1 with a marker identifying breast glandular cells, which have high tissue-IHC staining (HPA tissue IHC: breast glandular cells, High). Choose separated fluorophores and consider a longer-wavelength RPA1 channel when shorter-wavelength tissue autofluorescence obscures signal (general IF practice). Permeabilise fixed cells to expose a nuclear antigen, titrating detergent against preservation of nuclear detail; no antibody-specific IF fixation or epitope-access evidence is supplied (UniProt P27694: nucleus; general IF practice). Compare a DNA counterstain, no-primary control, and single-color controls before assigning nuclear puncta or apparent overlap to RPA1 (general IF practice).
What should I check when DAB color obscures RPA1 nuclei?
First inspect a no-primary control and review whether endogenous peroxidase was blocked before HRP detection (general chromogenic IHC practice). The selected image used a peroxidase-conjugated secondary antibody and DAB, while its 10% goat-serum block and 1:50 primary dilution are documented antibody conditions (caption M01317-1). If control sections remain colored, optimize the peroxidase block and wash steps before changing primary concentration (general IHC practice). If background appears only with primary antibody, titrate it and assess nonspecific edge or necrotic staining separately from intact nuclei (general IHC practice). Expected RPA1 staining is nuclear (HPA tissue IHC: general nuclear expression).
How should I quantify heterogeneous nuclear RPA1 staining in paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define positive staining within intact nuclei and keep the intensity threshold and scoring rules fixed across cases (HPA tissue IHC: general nuclear expression; general IHC practice). Report the percentage of positive nuclei and, when intensity matters, an H-score derived from the proportions at each staining grade (general IHC practice). For spatial comparisons, report positive nuclei per mm² of evaluable tissue and normalize counts to total evaluable nuclei or tissue area (general IHC practice). Score comparable cell compartments separately, exclude necrosis and section edges, and record the same sampling strategy for every specimen (general IHC practice).
How can I distinguish genuine RPA1 staining from IHC artefact?
A credible positive pattern is nuclear, consistent with general nuclear tissue expression and nucleoplasmic localisation (HPA tissue IHC; HPA subcellular: nucleoplasm). Check whether intact cells in the chosen compartment stain reproducibly across comparable areas; breast glandular cells are one documented high-staining reference (HPA tissue IHC: breast glandular cells, High). Treat color confined to section edges, necrotic regions, or nuclei with poor preservation as suspect, and inspect a no-primary control for endogenous enzyme signal (general IHC practice). Nuclear foci alone do not prove DNA damage or alternative telomere maintenance, even though RPA1 participates in DNA repair and can enrich in PML bodies under the stated condition (UniProt P27694: function and subcellular location).
Boster reagents
Best RPA1 / Replication protein A 70 kDa DNA-binding subunit IHC Antibodies
Anti-RPA1 antibodies have IHC images from human paraffin sections (catalog image captions) and an IF image from A549 cells (PB9886 image caption); catalog reactivity also lists mouse and monkey for M01317-2.
Real IHC data
Anti-RPA70 Monoclonal Antibody
Cat # M01317-1
Real IHC data
Anti-RPA70 RPA1 Antibody ® (monoclonal, 11H4)
Cat # M01317-2
Real IF data
Anti-RPA70/RPA1 Antibody ®
Cat # PB9886
M01317-1 has IHC images from human breast cancer, colon cancer and colon paraffin sections (M01317-1 image captions); M01317-2 has IHC images from human intestinal and lung cancer paraffin sections (M01317-2 image captions). PB9886 has an IF image from A549 cells and lists IF/ICC applications, with no IHC image or listed IHC application (PB9886 image caption and catalog applications).
Which to pick: For tissue IHC, choose rabbit monoclonal M01317-1 or mouse monoclonal M01317-2; each has its own paraffin-section IHC images (catalog hosts, clones and IHC image captions). For IF/ICC, PB9886 lists both applications and has an A549 IF image (PB9886 catalog applications and image caption). For mouse or monkey samples, M01317-2 lists those species and IHC, but its IHC images show human paraffin sections; the captions do not report the fixative (M01317-2 catalog reactivity, applications and IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P27694 (RFA1_HUMAN, Replication protein A 70 kDa DNA-binding subunit).
- Human Protein Atlas. RPA1 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. RPA1 subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. RPA1 antibody validation summary (3 antibodies).
- Unravelling the clinicopathological and functional significance of replication protein A (RPA) heterotrimeric complex in breast cancers. NPJ breast cancer 2023 — PMC10063624.
- Role and mechanism of RPA1 in the development and progression of glioma. Experimental and therapeutic medicine 2025 — PMC12235235.
- Clinicopathological and functional evaluation of replication protein A in epithelial ovarian cancers: A target validation study. Translational oncology 2026 — PMC12925576.
- Identification of potential biomarkers of head and neck squamous cell carcinoma using iTRAQ based quantitative proteomic approach. Data in brief 2018 — PMC6139602.
- PubMed PMID:2050703 — UniProt-cited evidence.
- PubMed PMID:8420996 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RPA1 (Replication protein A 70 kDa DNA-binding subunit). Expected localisation General nuclear staining in tissue (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M01317-1). Validated IHC antibody: M01317-1 at 1:50 (the product's own IHC validation image is shown). Controls (HPA): positive Adipose tissue (Adipocytes); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet M01317-1); 2. Fixation: Image fixative and duration unreported (datasheet M01317-1); 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 M01317-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M01317-1); 8. Primary antibody: Rabbit monoclonal (clone BB-18) anti-RPA1, 1:50 (datasheet M01317-1); 9. Primary incubation: Overnight at 4 °C (datasheet M01317-1). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: DNA damage may produce nuclear repair foci (UniProt); Isoform / epitope: No isoforms annotated; two chains differ at the N terminus (UniProt); Regulation: No tissue-specific regulation annotated (UniProt). Evidence: UniProt P27694, HPA tissue IHC · Supported, HPA ICC-IF · nucleoplasm., Datasheet M01317-1, PMC10063624, PMC12235235. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RPA1/immunohistochemistry-ihc-guide.