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RPA3 / Replication protein A 14 kDa subunit · IHC design guide
Design Immunohistochemistry for RPA3
Plan paraffin-section RPA3 staining with an IHC-validated antibody (datasheet A04696). This guide covers the nuclear and cytoplasmic tissue pattern, high staining in bone marrow hematopoietic cells, and the caveat of presumed off-target binding (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RPA3 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 (UniProt); nuclear and cytoplasmic in tissue (HPA tissue IHC) | |
| Staining pattern | Nuclear and cytoplasmic; high in hematopoietic cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A04696) | |
| 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 paraffin-section fixation consistent across specimens. (selected-SKU IHC image A04696) | |
| Caveat | Presumed off-target staining may confound interpretation (HPA tissue IHC) | |
| Regulation | No expression regulator annotated (UniProt) | |
| Isoform / epitope | 0 annotated isoforms; chain 2–121 (UniProt) |
Section 1
Recommended RPA3 IHC & IF Protocols
The catalog antibody uses heat-mediated Tris-EDTA retrieval at pH 9.0 (datasheet A04696). Two published RPA3 IHC protocols follow (PMC5661258; PMC10063624).
| Sample | Paraffin-embedded human oophoroma tissue; fixative not specified (datasheet A04696) |
| Fixation | Image fixative and duration unreported (datasheet A04696); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Tris-EDTA pH 9.0 (datasheet A04696); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-RPA3, 1:50 recommended; image 1:200 (datasheet A04696) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | RPA3-positive staining in adipocytes of adipose tissue (HPA tissue IHC: High). HPA tissue profile: General nuclear and cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 for the catalog antibody (datasheet A04696). The breast tissue protocol provides a citrate pH 6.0 comparison (PMC10063624).
Section 2
What Is the Expected RPA3 Staining Pattern?
RPA3 is a nuclear protein without a transmembrane segment (UniProt P35244: subcellular location and topology). In tissue IHC, staining may appear in nuclei and cytoplasm across many cell types (HPA: general nuclear and cytoplasmic expression; low tissue specificity). HPA rates its tissue IHC evidence Approved, with medium consistency between staining and RNA expression; presumed off-target binding was observed and disregarded (HPA: reliability description).
| Nuclear staining in hematopoietic cells of bone marrow or respiratory epithelial cells of bronchus. | This fits RPA3's nuclear location and the high staining reported for those cells (UniProt P35244: nucleus; HPA: High in bone marrow hematopoietic cells and bronchus respiratory epithelial cells). Compare intensity within each tissue rather than assuming every cell should stain equally. |
| Nuclear staining accompanied by cytoplasmic staining in the same section. | A cytoplasmic component is compatible with the reported tissue IHC profile, even though UniProt lists the nucleus as RPA3's location (HPA: general nuclear and cytoplasmic expression; UniProt P35244: nucleus). Record both compartments and assess whether the nuclear signal remains interpretable. |
| Signal confined to cell borders or extracellular material, with no convincing nuclear staining. | Treat this as a suspect compartment pattern: RPA3 is nuclear and has no transmembrane segment (UniProt P35244: location and topology). Check morphology and controls before calling the section positive; HPA's reported cytoplasmic staining does not establish a membrane or extracellular pattern (HPA: tissue IHC profile). |
| Unexpected staining in a cell population, especially where the control also develops color. | Consider nonspecific antibody binding or endogenous detection activity (general IHC practice). HPA reports low tissue specificity and supplies no negative cell list, so cell identity alone cannot establish a false positive (HPA: RNA specificity and tissue IHC record). Resolve the control result before assigning RPA3 expression. |
| Uniform haze across cells and surrounding tissue, or no signal in a high-staining reference tissue. | Haze can obscure the compartment needed for interpretation; absent signal in bone marrow hematopoietic cells or bronchus respiratory epithelial cells warrants a technical check (general IHC practice; HPA: High in those cells). Neither appearance alone establishes RPA3 absence or a target-specific fixation effect. |
💡Expected RPA3 appearanceCall a slide convincingly positive when interpretable nuclear staining appears in the relevant cells, with possible cytoplasmic staining; high staining is reported in selected cells such as bone marrow hematopoietic cells (UniProt P35244: nucleus; HPA: tissue IHC profile and High in bone marrow). Isolated border, extracellular, or control-matched staining is suspect (general IHC interpretation).
| Location and topology | UniProt places RPA3 in the nucleus and lists no transmembrane segment (UniProt P35244: location and topology). Use nuclear signal as the principal location check while allowing the cytoplasmic component reported in tissue IHC (HPA: tissue IHC profile). |
| Cell and tissue context | HPA reports low tissue specificity and high staining in adipocytes, glandular cells, hematopoietic cells, respiratory epithelial cells, glial cells, and cerebellar granular-layer cells (HPA: tissue IHC record). These are reference observations, not an exhaustive positive or negative cell list. |
| Antibody evidence | The listed rabbit polyclonal antibody HPA005708 is Approved for IHC, while the tissue summary notes medium staining–RNA consistency and disregarded presumed off-target binding (HPA: antibody status and reliability description). Interpret weak or atypical staining with controls. |
| IF/ICC Q: Is its staining pattern established here? | UniProt supports a nuclear expectation, but HPA provides no main ICC-IF location, image-bearing cell lines, or ICC validation status for the listed antibody (UniProt P35244: nucleus; HPA: subcellular and antibody records). A specific IF/ICC appearance is therefore unconfirmed by these payloads. |
| Fixation sensitivity | Target-specific fixation sensitivity is unreported in the supplied UniProt and HPA records (UniProt P35244; HPA: tissue IHC and subcellular records). Do not infer a fixation effect from topology, modifications, or tissue staining intensity. |
| Situation | Likely cause | Next action |
|---|---|---|
| No staining in a reference section expected to stain strongly. | The assay or reference section may have failed; high staining is reported for bone marrow hematopoietic cells (HPA: tissue IHC record). | Check section quality, retrieval conditions, primary-antibody application, detection reagents, and a concurrent positive control (general IHC practice). Do not assign a target-specific fixation cause without evidence. |
| Diffuse color makes nuclei hard to distinguish. | Background from blocking, antibody exposure, washing, or detection can obscure localisation (general IHC practice). | Review the no-primary control and adjust blocking, antibody concentration, washing, or chromogen development as indicated by that control (general IHC practice). Reassess nuclear and cytoplasmic signal separately. |
| Color is restricted to borders or extracellular deposits. | That distribution conflicts with UniProt's nuclear location and lack of a transmembrane segment (UniProt P35244: location and topology). | Inspect tissue morphology and negative controls, then reassess with the IHC-validated antibody and its established IHC conditions (general IHC practice; HPA: HPA005708 IHC Approved). |
| An unexpected cell population stains. | The result could reflect broad expression, nonspecific binding, or endogenous detection activity (HPA: low tissue specificity; general IHC practice). | Compare compartment and intensity with the same section's expected cells and the no-primary control. Avoid calling the population negative solely because it is absent from HPA's short positive list (HPA: tissue IHC record). |
| Cytoplasmic staining appears stronger than nuclear staining. | HPA reports both compartments, whereas UniProt specifies a nuclear location (HPA: tissue IHC profile; UniProt P35244: nucleus). The records do not define an acceptable nuclear-to-cytoplasmic ratio. | Document each compartment, review background controls, and reserve a confident localisation call for signal that remains distinguishable from background (general IHC interpretation). |
| An IF/ICC result is being compared with this chromogenic IHC guide. | HPA supplies no ICC-IF images, main location, or ICC validation status for the listed antibody (HPA: subcellular and antibody records). | Treat the tissue IHC pattern as context only. Interpret IF/ICC with its own controls and guide; do not transfer IHC validation to ICC-IF (general assay-validation practice). |
Experiment evidence
IHC & IF images showing RPA3 staining
A04696 · IHCImmunohistochemical analysis of paraffin-embedded human oophoroma. 1, Antibody was diluted at 1:200 (4° overnight). 2, Tris-EDTA, pH9.0 was
A04696-2 · IHCIHC analysis of RPA3 using anti-RPA3 antibody (A04696-2). RPA3 was detected in a paraffin-embedded section of human lung cancer tissue. Heat
A04696-1 · IFImmunofluorescence analysis of MCF7 cells using RPA3 antibody .
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000106399-RPA3/tissue/cerebral+cortex#imid_2258172
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000106399-RPA3/tissue/colon#imid_2258098
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000106399-RPA3/tissue/liver#imid_2258099
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000106399-RPA3/tissue/kidney#imid_2258108
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RPA3 IHC & IF
🧪Run bone marrow first and assess nuclear staining in hematopoietic cells (HPA: High in bone marrow hematopoietic cells; UniProt P35244: Nucleus). HPA detects RPA3 in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and treat cells without nuclear chromogen on the positive slide as a background comparison, not as confirmed RPA3-negative cells (HPA: no negative rows; detected in all 45 scored tissues).
Positive control tissue: Adipose tissue (Adipocytes, HPA High)
Negative control tissue: None in HPA: RPA3 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 carries no ICC-IF cell line for RPA3; derive a cell-line control from the positive tissue's cell type (Adipocytes) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only section, an isotype control matched to the primary antibody’s host species and clonality, and, if available, RPA3-knockout material as a biological specificity control (standard IHC practice). Quench endogenous peroxidase in bone marrow before chromogenic detection because hematopoietic cells can contain peroxidase activity (standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence; the selected A04696 paraffin-section caption does not state a fixative (selected-SKU tissue-IHC caption: fixative unreported). That caption uses Tris-EDTA at pH 9.0 for retrieval and a 1:200 primary dilution overnight at 4°C, but does not establish whether retrieval is required under other conditions (selected-SKU tissue-IHC caption). The supplied evidence does not establish that frozen sections or IF/ICC are easier, and bone marrow’s endogenous peroxidase requires attention when interpreting chromogenic signal (HPA subcellular: no ICC-IF images; standard IHC practice).
HPA tissue IHC evidence for RPA3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Presumed off target binding observed and disregarded.). 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 | Adipocytes | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: RPA3 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced RPA3 IHC Tips
Troubleshoot RPA3 staining in paraffin sections by checking retrieval, nuclear localisation, controls and scoring before interpreting chromogenic signal.
What should I change when nuclear RPA3 staining is weak after retrieval?
Start with heat-mediated Tris-EDTA retrieval at pH 9.0 for paraffin sections (datasheet A04696). The documented tissue image used primary antibody at 1:200 overnight at 4°C, so reproduce those conditions before attributing weak staining to retrieval (datasheet A04696). If staining remains weak, compare a cautiously adjusted heating time or a different retrieval buffer on adjacent sections while keeping detection conditions constant (standard IHC practice). Include a known staining control and a no-primary control in each run; excessive retrieval can damage section morphology and make apparent gains in signal hard to interpret (standard IHC practice).
Could fixation explain weak or uneven RPA3 staining?
Target-specific fixation sensitivity is unknown: the selected RPA3 image identifies paraffin-embedded tissue but does not state its fixative (datasheet A04696). Record the fixative, fixation duration and processing history for each specimen, then compare similarly processed sections before changing the antibody dilution (standard IHC practice). Assess section integrity and nuclear morphology alongside staining, because poor preservation can complicate interpretation of a nuclear target (UniProt P35244; standard IHC practice). If specimens differ in fixation history, test them with the same pH 9.0 Tris-EDTA retrieval and staining run before assigning differences to RPA3 abundance (datasheet A04696; standard IHC practice).
How should I evaluate nuclear and cytoplasmic RPA3 staining?
Score nuclear staining separately because RPA3 is assigned to the nucleus and functions in DNA replication and damage responses (UniProt P35244). Cytoplasmic staining needs cautious review: tissue IHC reports general nuclear and cytoplasmic expression, but its Approved assessment notes presumed off-target binding (HPA tissue IHC). Compare compartment patterns across intact cells, an appropriate positive control and a no-primary control, rather than treating all brown signal as equivalent (standard IHC practice). Document nuclear and cytoplasmic results separately, and require reproducible cell-associated staining before interpreting a cytoplasmic pattern as RPA3-related (UniProt P35244; HPA tissue IHC; standard IHC practice).
Could an isoform or epitope difference explain inconsistent staining?
The supplied RPA3 record lists 0 isoforms and no transmembrane segment, so an annotated isoform switch does not explain discordant section staining (UniProt P35244). Its chain spans residues 2–121, with N-terminal acetylation at residue 2; the payload does not identify this antibody’s epitope (UniProt P35244; datasheet A04696). Compare retrieval and primary antibody conditions on adjacent sections before proposing epitope masking, and ask for epitope information if that question becomes critical (standard IHC practice). Because the epitope is unspecified, neither its accessibility after processing nor an effect of acetylation can be assigned from these records (UniProt P35244; datasheet A04696).
How can IF help assess a questionable chromogenic RPA3 pattern?
Use IF as a separate comparison and first establish whether the antibody performs under the chosen IF conditions; the supplied product image documents paraffin-section IHC (datasheet A04696). Multiplex RPA3 with a marker for the expected cell population, such as adipocytes or hematopoietic cells when examining those tissues, and compare nuclear signals cell by cell (HPA tissue IHC; UniProt P35244). Select fluorophores outside the strongest tissue autofluorescence and include single-channel controls to assess bleed-through (standard IF practice). For this nuclear, non-transmembrane protein, optimise permeabilisation for access to an intracellular epitope, then check nuclear counterstain and no-primary controls (UniProt P35244; standard IF practice).
What should I check when the section has diffuse brown staining?
Inspect a no-primary control and the tissue morphology before changing the RPA3 interpretation; endogenous pigment, enzyme activity and nonspecific detection can produce chromogenic signal (standard IHC practice). Apply a peroxidase block for peroxidase-based detection and assess the blocking and wash steps used in the run (standard IHC practice). The selected image used primary antibody at 1:200 overnight at 4°C and secondary antibody at 1:200 for 45 minutes at room temperature (datasheet A04696). If background persists, compare antibody titrations against control sections while preserving the documented pH 9.0 retrieval as the starting condition (datasheet A04696; standard IHC practice).
How should I quantify RPA3 across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and nuclear scoring rules before reviewing groups, because RPA3 has a nuclear assignment while tissue IHC also reports cytoplasmic staining (UniProt P35244; HPA tissue IHC). Record the percentage of positive nuclei and staining intensity, then calculate an H-score if intensity grading is reproducible; keep cytoplasmic scores separate (standard IHC practice). For spatial comparisons, report positive-cell density per mm² of viable, analysable tissue and exclude folds, necrosis and section edges by preset criteria (standard IHC practice). Normalise to the number of eligible nuclei or assessed tissue area, and use matched processing, retrieval and detection settings across specimens (standard IHC practice).
When is an apparent RPA3-positive cell likely to be an artefact?
Give greatest weight to reproducible staining in intact nuclei, consistent with RPA3’s nuclear localisation and role in DNA metabolism (UniProt P35244). Compare the stained cell type with the specimen: high staining is reported in adipocytes and bone-marrow hematopoietic cells, but those observations do not establish specificity in every sample (HPA tissue IHC). Treat staining restricted to cut edges, necrotic areas or damaged cells as suspect, and check a no-primary control for detection-related signal (standard IHC practice). Diffuse cytoplasmic or unexpected-cell staining requires independent confirmation because the tissue IHC assessment explicitly notes presumed off-target binding (HPA tissue IHC).
Boster reagents
Best RPA3 / Replication protein A 14 kDa subunit IHC Antibodies
Anti-RPA3 antibodies have IHC images from human paraffin sections and an IF image from MCF7 cells (catalog image captions); listed reactivity extends to mouse and rat, depending on SKU (catalog reactivity).
Real IHC data
Anti-RPA14 RPA3 Antibody
Cat # A04696
Real IHC data
Anti-RPA3 Antibody ®
Cat # A04696-2
Real IF data
Anti-RPA3 Antibody
Cat # A04696-1
A04696 has an IHC image from human oophoroma in a paraffin section; A04696-2 has one from a paraffin section of human lung cancer (respective IHC image captions). A04696-1 is listed for IF/ICC and has an IF image of MCF7 cells (catalog applications; A04696-1 IF image caption).
Which to pick: For tissue IHC, choose A04696 for listed human and mouse reactivity, or A04696-2 for human samples; both are polyclonal and have human paraffin-section IHC images (catalog reactivity and dilution_raw; respective IHC image captions). For IF/ICC, choose A04696-1, which lists human, mouse and rat reactivity and shows IF in MCF7 cells; its clonality is unreported (catalog applications, reactivity and clone; A04696-1 IF image caption). For cross-species IHC, A04696 lists human and mouse, while its paraffin-section image documents human tissue only; neither IHC caption reports a fixative (catalog reactivity; respective IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P35244 (RFA3_HUMAN, Replication protein A 14 kDa subunit).
- Human Protein Atlas. RPA3 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. RPA3 subcellular location (ICC-IF): Highest expression in GAMG: 172.0 nTPM.
- Human Protein Atlas. RPA3 antibody validation summary (1 antibodies).
- RPA3 is a potential marker of prognosis and radioresistance for nasopharyngeal carcinoma. Journal of cellular and molecular medicine 2017 — PMC5661258.
- Unravelling the clinicopathological and functional significance of replication protein A (RPA) heterotrimeric complex in breast cancers. NPJ breast cancer 2023 — PMC10063624.
- Clinicopathological and functional evaluation of replication protein A in epithelial ovarian cancers: A target validation study. Translational oncology 2026 — PMC12925576.
- PubMed PMID:8454588 — UniProt-cited evidence.
- PubMed PMID:12853948 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RPA3 (Replication protein A 14 kDa subunit). Expected localisation Nuclear (UniProt); nuclear and cytoplasmic in tissue (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A04696). Validated IHC antibody: A04696 at 1:50 recommended; image 1:200 (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 workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human oophoroma tissue; fixative not specified (datasheet A04696); 2. Fixation: Image fixative and duration unreported (datasheet A04696); 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: Tris-EDTA pH 9.0 (datasheet A04696); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-RPA3, 1:50 recommended; image 1:200 (datasheet A04696); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep paraffin-section fixation consistent across specimens. (selected-SKU IHC image A04696); Caveat: Presumed off-target staining may confound interpretation (HPA tissue IHC); Isoform / epitope: 0 annotated isoforms; chain 2–121 (UniProt); Regulation: No expression regulator annotated (UniProt). Evidence: UniProt P35244, HPA tissue IHC · Approved, HPA cell-line RNA · Highest expression in GAMG: 172.0 nTPM, Datasheet A04696, PMC5661258, PMC10063624. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RPA3/immunohistochemistry-ihc-guide.