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RAD51B / DNA repair protein RAD51 homolog 2 · IHC design guide
Design Immunohistochemistry for RAD51B
Plan RAD51B staining in paraffin sections using its general nuclear tissue profile (HPA tissue IHC). This guide covers fixation, detection and interpretation, with an IHC-validated antibody diluted 1:100–1:300 (datasheet: A20019).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RAD51B 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 (HPA tissue IHC) | |
| Staining pattern | Nuclei of glandular, hematopoietic and neuronal cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | None in HPA (detected in all 44 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Antibody staining has medium consistency with RNA data (HPA tissue IHC) | |
| Regulation | DNA damage role; expression response unknown (UniProt) | |
| Isoform / epitope | 5 isoforms; check epitope coverage; no membrane topology (UniProt) |
Section 1
Recommended RAD51B IHC & IF Protocols
The catalog antibody IHC-P protocol is accompanied by published RAD51B paraffin-section workflows (PMC4016303; PMC12827460).
| Sample | Paraffin-embedded human pancreas tissue; fixative not specified (datasheet A20019) |
| Fixation | Image fixative and duration unreported (datasheet A20019); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear antigen) |
| 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-RAD51B, 1:100 - 1:300 (datasheet A20019) |
| 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 | RAD51B-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0, 95–98 °C, 20 min (page antigen retrieval); citrate pH 6.0 is a published alternative (PMC4016303).
Section 2
What Is the Expected RAD51B Staining Pattern?
RAD51B is a nuclear DNA repair protein with no annotated transmembrane segment (UniProt O15315: subcellular location and topology). In paraffin sections, expect predominantly nuclear staining across several cell types: HPA describes general nuclear expression and reports high staining in selected glandular, hematopoietic, respiratory epithelial, and neuronal cells (HPA: tissue IHC). HPA rates its tissue IHC profile Enhanced, while describing only medium consistency between antibody staining and RNA expression (HPA: tissue IHC reliability).
| Nuclear signal in expected cells, with limited staining outside nuclei. | This agrees with RAD51B nuclear localization (UniProt O15315) and HPA's general nuclear IHC profile (HPA: tissue IHC). High staining is reported in breast and appendix glandular cells, bone marrow hematopoietic cells, bronchial respiratory epithelial cells, and cerebral cortex neurons (HPA: tissue IHC). Compare cells within the same section before judging intensity (standard IHC practice). |
| Predominantly cytoplasmic, membranous, or extracellular stain. | These compartments disagree with the expected nuclear pattern (UniProt O15315: nucleus; HPA: tissue IHC). Review morphology and the negative reagent control before accepting the signal as RAD51B; nonspecific binding or detection background can produce misplaced chromogen (standard IHC practice). A small apparent rim around crowded nuclei needs careful microscopic review (standard IHC practice). |
| Strong stain in a cell population while the expected cells in that section lack nuclear signal. | Consider cross-reactivity or endogenous detection activity, especially if chromogen appears outside nuclei (standard IHC practice). RAD51B is broadly expressed, and HPA supplies no verified negative tissue in this payload, so an unlisted cell type alone cannot establish a false positive (UniProt O15315: tissue specificity; HPA: tissue IHC). |
| Diffuse chromogen over nuclei, cytoplasm, and surrounding tissue. | Widespread haze obscures the nuclear pattern and weakens interpretation (HPA: general nuclear IHC profile; standard IHC practice). Compare a no-primary control for detection background, then review blocking, washing, antibody concentration, and chromogen development as general IHC variables (standard IHC practice). |
| No nuclear signal in a reported high-staining tissue. | A blank result in bone marrow hematopoietic cells or breast glandular cells conflicts with HPA's reported high staining, but does not by itself prove target absence (HPA: tissue IHC). Check control staining, tissue preservation, retrieval, reagent performance, and detection settings using the assay's validated workflow (standard IHC practice). |
💡Expected RAD51B appearanceCall positive when interpretable cells show predominantly nuclear chromogen, including strong signal in HPA-reported high-staining populations; dominant cytoplasmic or tissue-wide haze warrants review as a possible false positive (HPA: tissue IHC; UniProt O15315: nucleus; standard IHC practice).
| Cell population and tissue | HPA reports High staining in adrenal, appendix, breast, and cervical glandular cells; bone marrow hematopoietic cells; bronchial respiratory epithelial cells; and caudate and cerebral cortex neurons (HPA: tissue IHC). These are reference observations, not guaranteed intensity in every specimen (standard IHC practice). |
| Antibody evidence | CAB016191 is mouse monoclonal and IHC Enhanced; HPA051869 is rabbit polyclonal with ICC Approved status and no IHC status in this payload (HPA: antibody validation). Keep assay-specific validation separate when comparing images or selecting an IHC antibody (standard IHC practice). |
| Isoforms and antigen identity | UniProt lists 5 RAD51B isoforms, but this payload gives no antibody epitope or isoform coverage (UniProt O15315; HPA: antibody validation). An unexpected pattern cannot be assigned to a particular isoform from these records alone. |
| Cell structure and processing | RAD51B is annotated as nuclear, with no transmembrane segment, signal peptide, or propeptide and one chain spanning residues 1–384 (UniProt O15315). These annotations support a nuclear readout; they provide no target-specific evidence about fixation or retrieval sensitivity. |
| IF/ICC appearance? | HPA ICC-IF places RAD51B mainly in nuclear bodies, with additional nucleoplasmic localization; images are listed for A-431, MCF-7, and U2OS (HPA: subcellular). This IF observation helps interpret localization, but does not define chromogenic IHC spot counts or intensity (standard assay interpretation). |
| Situation | Likely cause | Next action |
|---|---|---|
| Nuclei are blank in a reported high-staining reference tissue. | Weak assay signal, tissue quality, or a missed detection step is possible; the record does not identify a RAD51B-specific failure mechanism (HPA: tissue IHC; standard IHC practice). | Confirm the reference cell population, inspect section integrity, and review the validated IHC-P workflow, reagent controls, and detection steps (HPA: tissue IHC; standard IHC practice). |
| Chromogen is strongest outside nuclei. | The distribution conflicts with nuclear RAD51B and may reflect nonspecific antibody binding or detection activity (UniProt O15315; HPA: tissue IHC; standard IHC practice). | Compare the no-primary control, inspect morphology, and optimize blocking and washing within the established IHC workflow (standard IHC practice). |
| All cell types and extracellular spaces show a brown haze. | General background can obscure compartment-specific interpretation (standard IHC practice). | Review antibody concentration, wash steps, and chromogen development against the validated protocol; retain a no-primary control for comparison (standard IHC practice). |
| A bright signal appears only in an unlisted cell type. | HPA reports broad nuclear expression and provides no negative tissue here; the isolated signal remains uncertain (HPA: tissue IHC; UniProt O15315: tissue specificity). | Check whether the signal is nuclear, compare expected cells in the same section, and assess reagent controls before calling cross-reactivity (HPA: tissue IHC; standard IHC practice). |
| Two antibodies yield different distributions. | Their listed evidence covers different applications: CAB016191 has IHC Enhanced status, while HPA051869 has ICC Approved status only (HPA: antibody validation). | Interpret each in its validated application; for paraffin IHC, prioritize IHC validation and compare nuclear localization with tissue controls (HPA: antibody validation and tissue IHC; standard IHC practice). |
| IF/ICC shows punctate nuclei while IHC looks more evenly nuclear. | HPA localizes RAD51B mainly to nuclear bodies and additionally to nucleoplasm by ICC-IF; tissue IHC is summarized as general nuclear expression (HPA: subcellular; HPA: tissue IHC). | Score each application against its reported pattern; do not require discrete IF-like puncta in chromogenic paraffin sections (HPA: subcellular and tissue IHC; standard assay interpretation). |
Experiment evidence
IHC & IF images showing RAD51B staining
A20019 · IHCImmunohistochemistry analysis of paraffin-embedded human pancreas tissue, using RAD51L1 Antibody. The picture on the right is blocked with t
A02658-2 · IFIF analysis of RAD51L1/RAD51B using anti-RAD51L1/RAD51B antibody (A02658-2) and anti-Beta Tubulin antibody (M01857-3). RAD51L1/RAD51B was de
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000182185-RAD51B/tissue/cerebral+cortex#imid_4330980
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000182185-RAD51B/tissue/colon#imid_4330906
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000182185-RAD51B/tissue/liver#imid_4330923
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000182185-RAD51B/tissue/kidney#imid_4330916
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA051869; green = RAD51B, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA051869; green = RAD51B, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RAD51B IHC & IF
🧪Run bone marrow first: hematopoietic cells should show nuclear staining (HPA: High in bone marrow hematopoietic cells; UniProt O15315: nucleus). HPA detects RAD51B in all 44 scored tissues, so there is no HPA-supported negative tissue; no-primary and isotype controls provide the negative reference, while cells without nuclear staining on the positive slide should show only background and should not be assumed to be target-negative (HPA: no negative tissue rows).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: RAD51B is detected in all 44 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 RAD51B in A-431, MCF-7, U2OS, with annotated localisation: Nuclear bodies (approved) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, an isotype control matched to the primary antibody’s host species and immunoglobulin class, and a verified RAD51B knockout sample as a biological negative (standard IHC practice). Quench endogenous peroxidase in bone marrow before chromogenic detection, particularly in myeloid cells (standard IHC practice).
⚠️Feasibility: No supplied target or application evidence reports a RAD51B-specific fixation window or fixation effect; the A20019 paraffin-section pancreas caption does not state a fixative (selected-SKU tissue-IHC caption: fixative unreported). Antigen retrieval dependence is unreported, so optimize retrieval on paraffin sections; the supplied evidence does not establish that frozen sections or IF are easier (selected-SKU tissue-IHC caption; HPA: ICC-IF images in A-431, MCF-7 and U2OS). For IF localisation checks, HPA reports nuclear bodies with additional nucleoplasmic staining; in bone marrow IHC, endogenous myeloid peroxidase can create chromogenic background (HPA: subcellular localisation; standard IHC practice).
HPA tissue IHC evidence for RAD51B
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — 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 |
|---|---|---|---|---|
| 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 → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: RAD51B is detected in all 44 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced RAD51B IHC Tips
Troubleshoot RAD51B staining by checking nuclear localisation, section processing, antibody specificity and cell-based scoring.
How should I retrieve RAD51B in paraffin sections when nuclear staining is weak?
Use Tris-EDTA at pH 9.0 for heat-induced epitope retrieval at 95–98 °C for 20 min (page retrieval rule: nuclear antigen). Allow sections to cool in retrieval buffer, then keep the antibody and detection conditions constant while assessing whether nuclear staining improves (standard IHC practice). If staining remains weak, test a milder retrieval condition on adjacent sections as a fallback, because excess heat can damage morphology (standard IHC practice). Score nuclei in intact cells rather than judging improvement from diffuse section colour; RAD51B is nuclear (UniProt O15315: subcellular location). Record the retrieval condition used for each slide so staining comparisons remain interpretable (standard IHC practice).
Could fixation explain weak or uneven RAD51B staining?
Target-specific fixation sensitivity is unknown from the supplied evidence, so first document the fixative and processing history of each specimen (supplied evidence: no RAD51B fixation study). The A20019 image shows staining in paraffin-embedded human pancreas, but its caption does not state the fixative (A20019 tissue-IHC caption). Compare similarly processed sections using the same retrieval and detection settings before attributing differences to biology (standard IHC practice). Inspect tissue integrity and staining at the section edge, where processing differences can complicate interpretation (standard IHC practice). Report the fixation history as unknown when it cannot be recovered, without inferring a RAD51B-specific effect from staining patterns (standard IHC practice).
Which staining pattern supports RAD51B localisation in chromogenic IHC?
Prioritise nuclear staining in intact cells: RAD51B is assigned to the nucleus (UniProt O15315: subcellular location), and tissue IHC shows general nuclear expression (HPA: tissue profile). Nuclear bodies are the main location in cell imaging, with additional nucleoplasmic localisation (HPA: subcellular summary). In chromogenic sections, assess whether signal follows nuclear contours against the counterstain rather than treating isolated dark deposits as discrete nuclear bodies (standard IHC practice). Review cytoplasmic or extracellular colour against a negative control before calling it RAD51B, since those patterns differ from the reported localisation (UniProt O15315: subcellular location; standard IHC practice). Preserve exposure and scoring criteria across sections being compared (standard IHC practice).
How can isoforms and epitope accessibility affect RAD51B IHC interpretation?
RAD51B has 5 annotated isoforms, so an antibody's recognised isoforms depend on its documented epitope rather than the gene name alone (UniProt O15315: isoforms; standard IHC practice). Check the catalog antibody's immunogen or epitope description before interpreting a negative nucleus as absence of every isoform (standard IHC practice). The supplied record annotates no transmembrane segment, glycosylation sites or modified residues, but those annotations do not establish epitope exposure in paraffin sections (UniProt O15315: topology and annotations; standard IHC practice). A20019 staining in paraffin-embedded pancreas includes a synthesized-peptide blocking comparison; assess that control alongside morphology without treating it as proof of isoform specificity (A20019 tissue-IHC caption; standard IHC practice).
How should I check RAD51B localisation when adding an IF multiplex readout?
Treat IF as a separate assay and compare its nuclear RAD51B signal with the chromogenic section pattern (UniProt O15315: subcellular location; standard assay practice). For a glandular-cell sample, multiplex with a suitable epithelial marker to identify the expected compartment; high RAD51B tissue staining is reported in glandular cells in several tissues (HPA: tissue IHC positives; standard IF practice). Choose a fluorophore in a channel with low sample autofluorescence, and include single-colour and unstained controls before interpreting overlap (standard IF practice). Permeabilise to permit antibody access to the nucleus; RAD51B has no annotated transmembrane segment, so membrane sidedness does not define its epitope access (UniProt O15315: topology; standard IF practice).
What should I troubleshoot when RAD51B DAB staining looks diffuse?
Compare diffuse colour with a section processed without primary antibody to assess detection-system background (standard IHC practice). Use a peroxidase block before chromogenic detection and inspect for endogenous pigment or enzyme activity that could resemble DAB signal (standard IHC practice). Reduce excessive antibody or detection exposure according to the reagent instructions, then recheck whether nuclear contrast improves without losing tissue detail (standard IHC practice). RAD51B is reported as nuclear, so diffuse extracellular deposits or uniform cytoplasmic colour need separate scrutiny (UniProt O15315: subcellular location; standard IHC practice). The A20019 peptide-blocked pancreas image provides a specificity comparison for that SKU, although it does not establish the cause of background in another specimen (A20019 tissue-IHC caption).
How should I score RAD51B staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define a nuclear-positive threshold against the negative control and apply it consistently to morphologically intact cells (UniProt O15315: subcellular location; standard IHC practice). Report the percentage of positive nuclei and, when intensity is reliable, an H-score calculated from the percentages at each intensity grade (standard IHC scoring practice). If counting discrete positive cells, report density per mm² of viable tissue and identify the cell population scored (standard IHC quantification practice). Normalise comparisons to the number of evaluable nuclei or viable tissue area, and exclude folds, edges and necrotic regions using the same rules for every section (standard IHC practice). Keep retrieval and detection settings matched across groups (standard IHC practice).
How can I distinguish true RAD51B positivity from staining artefact?
Look for reproducible staining within intact nuclei, consistent with RAD51B's nuclear assignment and the general nuclear tissue pattern (UniProt O15315: subcellular location; HPA: tissue profile). Check the relevant cell population against morphology rather than assigning signal to nearby cells; HPA reports high staining in specified glandular, hematopoietic, respiratory epithelial and neuronal populations (HPA: tissue IHC positives). Disregard isolated edge staining, necrotic debris and colour also present without primary antibody until processing or detection background has been assessed (standard IHC practice). Endogenous peroxidase can confound DAB interpretation, so review the peroxidase-block control and tissue context (standard IHC practice). The A20019 peptide-blocked pancreas comparison supports evaluation of that antibody's staining pattern, without establishing specificity in every specimen (A20019 tissue-IHC caption).
Boster reagents
Best RAD51B / DNA repair protein RAD51 homolog 2 IHC Antibodies
A20019 has IHC data from paraffin-embedded human pancreas (A20019 figure) and lists human, monkey and mouse reactivity (catalog); A02658-2 has IF data from human U2OS cells (A02658-2 figure).
Real IHC data
Anti-RAD51L1 RAD51B Antibody
Cat # A20019
Real IF data
Anti-RAD51L1/RAD51B Antibody ®
Cat # A02658-2
A20019 will render with a paraffin-embedded human pancreas IHC image that includes a peptide-blocked comparison (A20019 figure). A02658-2 will render with an IF image of U2OS cells and lists human reactivity (A02658-2 figure; catalog).
Which to pick: Choose A20019 for tissue IHC: it is listed for IHC, is polyclonal, and has a paraffin-embedded human pancreas image (catalog; A20019 figure); its fixative is unreported (A20019 figure). Choose A02658-2 for human IF/ICC because both applications are listed and its U2OS IF image documents cell staining (catalog; A02658-2 figure). For cross-species work, A20019 lists human, monkey and mouse reactivity, while A02658-2 lists human only (catalog); A20019’s displayed IHC evidence is from human tissue (A20019 figure).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O15315 (RA51B_HUMAN, DNA repair protein RAD51 homolog 2).
- Human Protein Atlas. RAD51B tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. RAD51B subcellular location (ICC-IF): Mainly localized to the nuclear bodies. In addition localized to the nucleoplasm..
- Human Protein Atlas. RAD51B antibody validation summary (2 antibodies).
- Germline mutation in the RAD51B gene confers predisposition to breast cancer. BMC cancer 2013 — PMC4016303.
- Sarcomas With RAD51B Fusions Are Associated With a Heterogeneous Phenotype. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 2024 — PMC11251009.
- Uterine PEComas: A Morphologic, Immunohistochemical, and Molecular Analysis of 32 Tumors. The American journal of surgical pathology 2018 — PMC6133752.
- RAD51B-EZH2 axis as a potential therapeutic target for TNBC through cell fate conversion. Cell death & disease 2025 — PMC12827460.
- PubMed PMID:9207106 — UniProt-cited evidence.
- PubMed PMID:9441753 — UniProt-cited evidence.
- PubMed PMID:9512535 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RAD51B (DNA repair protein RAD51 homolog 2). Expected localisation General nuclear staining (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A20019 at 1:100 - 1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative None in HPA (detected in all 44 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human pancreas tissue; fixative not specified (datasheet A20019); 2. Fixation: Image fixative and duration unreported (datasheet A20019); 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-induced epitope retrieval in Tris-EDTA buffer, pH 9.0 , 20 min at 95–98 °C…; 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-RAD51B, 1:100 - 1:300 (datasheet A20019); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Antibody staining has medium consistency with RNA data (HPA tissue IHC); Isoform / epitope: 5 isoforms; check epitope coverage; no membrane topology (UniProt); Regulation: DNA damage role; expression response unknown (UniProt). Evidence: UniProt O15315, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nuclear bodies. In addition localized to the…, Datasheet A20019, PMC4016303, PMC12827460. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RAD51B/immunohistochemistry-ihc-guide.