This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
RAD23B / Lysine-specific demethylase RAD23B · IHC design guide
Design Immunohistochemistry for RAD23B
Plan RAD23B paraffin IHC using the catalog antibody at 1:100–1:300 (datasheet A02174-2). Assess nuclear and cytoplasmic staining (HPA tissue IHC) while accounting for cell cycle dependent localization (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RAD23B 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 staining across tissues (HPA tissue IHC) | |
| Staining pattern | Glandular cells: high nuclear and cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 8.0 HIER, heat-mediated (datasheet A02174-2) | |
| 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 | Cell cycle can shift the nuclear/cytoplasmic balance (UniProt) | |
| Regulation | Nuclear levels decrease in S phase (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope differences are unspecified (UniProt) |
Section 1
Recommended RAD23B IHC & IF Protocols
The catalog antibody protocol uses Tris-EDTA pH 8.0 retrieval (datasheet A02174-2). Published IHC protocols provide colorectal tissue and human brain examples (PMC12573188; PMC13140639).
| Sample | Paraffin-embedded Human brain tissue; fixative not specified (datasheet A02174-2) |
| Fixation | Image fixative and duration unreported (datasheet A02174-2); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Tris-EDTA pH 8.0 (datasheet A02174-2); 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-RAD23B, 1:100-1:300 (datasheet A02174-2) |
| 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 | RAD23B-positive staining in adipocytes of adipose tissue (HPA tissue IHC: High). HPA tissue profile: Nuclear and cytoplasmic expression in all tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated Tris-EDTA pH 8.0 for the catalog antibody (datasheet A02174-2); the published protocols used citrate retrieval (PMC12573188; PMC13140639).
Section 2
What Is the Expected RAD23B Staining Pattern?
RAD23B should show nuclear and cytoplasmic staining across tissues (HPA tissue IHC: nuclear and cytoplasmic expression in all tissues). Nucleoplasmic signal is supported by ICC-IF, with additional cytosolic signal (HPA subcellular: supported). Interpretation should allow both compartments because distribution changes with the cell cycle (UniProt P54727: subcellular location). RAD23B has no transmembrane segment, so a membrane-restricted pattern is unexpected (UniProt P54727: topology). HPA rates the tissue IHC profile Enhanced, while reporting medium consistency with RNA data (HPA tissue IHC: reliability).
| Nuclear staining with some cytoplasmic staining in tissue cells. | This fits the reported distribution (HPA tissue IHC: nuclear and cytoplasmic expression in all tissues). Differences between individual cells can be plausible: RAD23B is nuclear and cytoplasmic in G1, nuclear levels decrease in S phase, and it moves into the cytoplasm during mitosis (UniProt P54727: subcellular location). |
| A sharp cell-surface rim is the dominant pattern, without convincing intracellular staining. | Treat this as a suspected artefact and review the control slide: RAD23B has no transmembrane segment (UniProt P54727: topology), and the observed tissue pattern is nuclear and cytoplasmic (HPA tissue IHC: profile). Cytoplasmic signal alone is less decisive because cell-cycle-dependent redistribution is reported (UniProt P54727: subcellular location). |
| Unexpectedly strong staining is confined to a cell population expected to stain weakly. | Consider cross-reactivity or endogenous detection activity, then compare cell identity and controls (general IHC practice). Hematopoietic cells in bone marrow are reported at a low level, but HPA lists no negative tissue or cell population here; low staining cannot serve as an absolute absence control (HPA tissue IHC: low; negative list empty). |
| Brown color spreads across tissue spaces or coats many cells without discernible nuclei. | This is difficult to score as RAD23B because the reported signal is intracellular (HPA tissue IHC: profile; UniProt P54727: subcellular location). Check reagent-only controls, wash quality and chromogen development for nonspecific background or endogenous detection activity (general chromogenic IHC practice). |
| No convincing staining appears in a tissue and cell population reported as high. | Adipocytes in adipose tissue and neuronal cells in cerebral cortex are examples reported as high (HPA tissue IHC: positive cells). Confirm that the expected cells are present, then review the IHC-validated antibody, detection controls and general retrieval workflow (general IHC practice); the supplied sources do not establish RAD23B-specific fixation sensitivity. |
💡Expected RAD23B appearanceCall the result positive when recognizable tissue cells show nuclear staining with permissible cytoplasmic signal, including high staining in an HPA-listed high cell population; a dominant membrane rim or acellular brown deposit is suspect (HPA tissue IHC: profile and positive cells; UniProt P54727: topology; general IHC practice).
| Cell cycle and compartment | Nuclear and cytoplasmic signal is reported in G1; nuclear levels fall in S phase, and mitotic RAD23B relocates to the cytoplasm without chromatin association (UniProt P54727: subcellular location). Interpret a mixed field cell by cell before calling a weaker nucleus abnormal. |
| Choice of tissue and cells | The tissue profile spans all tissues, but reported levels differ by cell type (HPA tissue IHC: profile and positive cells). HPA lists high staining in adipocytes, glandular cells, respiratory epithelial cells and selected neural cells, while bone-marrow hematopoietic cells are low (HPA tissue IHC: positive and low cells). |
| IHC evidence strength | The tissue profile is rated Enhanced, with medium consistency between antibody staining and RNA expression (HPA tissue IHC: reliability description). Two listed antibodies have Enhanced IHC validation and one has Supported IHC validation (HPA antibodies: IHC status); assess the actual slide and controls rather than treating validation as proof of every stained structure. |
| Topology and processing | RAD23B has no transmembrane segment, signal peptide or propeptide; the listed chain spans residues 1–409 (UniProt P54727: topology and processing). These annotations support an intracellular interpretation but do not identify the antibody epitope or predict an antigen retrieval condition. |
| Isoforms and epitope uncertainty | Two isoforms are listed (UniProt P54727: isoforms). Their existence alone does not show which form the IHC-validated antibody detects; no epitope location is supplied. Avoid assigning a compartment difference or a missing signal to isoform selectivity without antibody-specific evidence. |
| IF/ICC comparison | Q: Should IF/ICC look identical to paraffin IHC? A: Use it as a compartment check: the main supported location is nucleoplasm, with additional cytosol (HPA subcellular: ICC-IF). The supplied IF record does not establish matching staining intensity or an IF/ICC protocol for this IHC guide. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected high-staining cells are blank. | The expected cell population may be absent from the section, or the IHC detection workflow may have failed (general IHC practice). | Locate an HPA-listed high cell population, then check the IHC-validated antibody and positive-control slide; review retrieval and detection steps as general IHC workflow checks (HPA tissue IHC: positive cells; general IHC practice). |
| Only a strong membrane rim is visible. | That compartment conflicts with the reported intracellular pattern and lack of a transmembrane segment (HPA tissue IHC: profile; UniProt P54727: topology). | Compare a control slide and examine whether nuclear or cytoplasmic staining is present in identifiable cells; investigate nonspecific edge staining before scoring the rim (general IHC practice). |
| Bone-marrow hematopoietic cells stain as strongly as chosen high controls. | HPA reports these cells as low, although it provides no negative cell population (HPA tissue IHC: low; negative list empty). | Recheck cell identity and use appropriate detection controls to assess cross-reactivity or endogenous activity; do not score low as necessarily zero (HPA tissue IHC: low; general IHC practice). |
| Staining varies markedly among neighboring nuclei. | Nuclear abundance can change during the cell cycle (UniProt P54727: subcellular location). | Inspect accompanying cytoplasmic signal and cell morphology before calling the variation a failure; compare the field with the reported nuclear and cytoplasmic tissue pattern (HPA tissue IHC: profile; UniProt P54727: subcellular location). |
| Diffuse brown haze obscures cellular boundaries. | Background can arise from detection activity, excess chromogen development or inadequate washing (general chromogenic IHC practice). | Review reagent-only controls and the detection and washing steps; score RAD23B only where intracellular staining can be assigned to recognizable cells (general IHC practice; HPA tissue IHC: profile). |
| IF/ICC shows cytosol as well as nucleoplasm. | Both locations are supported by ICC-IF, and distribution can vary with cell cycle (HPA subcellular: supported locations; UniProt P54727: subcellular location). | Treat cytosolic signal as potentially consistent with RAD23B when cellular staining is interpretable; use the separate IF/ICC guide for IF-specific workflow decisions (HPA subcellular: supported locations). |
Experiment evidence
IHC & IF images showing RAD23B staining
A02174-2 · IHCImmunohistochemical analysis of paraffin-embedded Human brain. Antibody was diluted at 1:100 (4° overnight). High-pressure and temperature T
M02174 · IHCImmunohistochemical analysis of paraffin-embedded Prostate Cancer using Rad23B mouse mAb (1/100 dilution).Antigen retrieval was performed by
M02174 · IFImmunocytochemistry staining of HeLa cells fixed with 4% Paraformaldehyde and using anti-Rad23B antibody (dilution 1:100).
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000119318-RAD23B/tissue/cerebral+cortex#imid_8172056
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000119318-RAD23B/tissue/colon#imid_8171982
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000119318-RAD23B/tissue/liver#imid_8171975
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000119318-RAD23B/tissue/kidney#imid_8171984
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA029718; green = RAD23B, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA029718; green = RAD23B, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RAD23B IHC & IF
🧪Run adipose tissue first: adipocytes should stain (HPA: High in adipocytes). HPA detects RAD23B in all 45 scored tissues, so use no-primary and isotype controls as negatives; no cell type on the positive slide is established as an internal negative, and apparently unstained cells should be treated only as background comparators (HPA: no negative tissue rows).
Positive control tissue: Adipose tissue (Adipocytes, HPA High)
Negative control tissue: None in HPA: RAD23B 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 RAD23B in A-431, U-251MG, U2OS, RPTEC/TERT1, hTERT-RPE1 (serum starved), with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a secondary-only (no-primary) section, a control matched to the primary antibody’s host species and clonality, and a knockout sample or peptide-preabsorbed antibody as a biological specificity control (caption: peptide-preabsorbed negative control). For chromogenic IHC, block endogenous peroxidase; in adipose tissue, distinguish staining in the thin adipocyte rim from empty lipid spaces (standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the A02174-2 paraffin-section caption does not state a fixative (caption: fixative unreported). That caption uses high-pressure, high-temperature Tris-EDTA retrieval at pH 8.0, but does not establish that retrieval is required for RAD23B staining (caption: retrieval conditions). Neither frozen sections nor IF can be judged easier from the supplied evidence; HPA reports nucleoplasmic and cytosolic ICC-IF localization, while adipocyte lipid spaces complicate interpretation of tissue staining (HPA: supported nucleoplasm and cytosol localization; standard IHC practice).
HPA tissue IHC evidence for RAD23B
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 |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular 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: RAD23B is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced RAD23B IHC Tips
Troubleshoot RAD23B staining in paraffin sections by checking retrieval, compartment patterns, controls and scoring before interpreting chromogenic signal.
What retrieval should I try first for weak RAD23B staining in paraffin sections?
Start with heat-mediated Tris-EDTA at pH 8.0 for antigen retrieval (datasheet A02174-2). The selected paraffin-section image used high-pressure, high-temperature retrieval in that buffer, followed by antibody at 1:100 overnight at 4°C (caption A02174-2). If staining remains weak, compare heating durations on adjacent sections while holding antibody concentration and detection conditions constant (standard IHC practice). Include a known positive section and a no-primary control so stronger retrieval is not mistaken for greater specificity (standard IHC practice). Judge improvement by nuclear and cytoplasmic staining in appropriate cells, rather than by overall brown intensity alone (HPA: tissue IHC profile).
Could fixation explain weak or uneven RAD23B staining?
Target-specific sensitivity to fixation is unknown from the supplied evidence; the selected paraffin-section caption does not state a fixative (caption A02174-2). Record the fixative, fixation duration and processing history for each specimen before comparing staining across batches (standard IHC practice). For a practical comparison, stain adjacent sections from differently processed specimens together using the same Tris-EDTA pH 8.0 retrieval and detection run (datasheet A02174-2; standard IHC practice). Check section adhesion, morphology and counterstain before changing antibody concentration, because damaged sections can produce patchy signal (standard IHC practice). Do not attribute a fixation effect to RAD23B without a controlled comparison (standard IHC practice).
How should I assess a mainly cytoplasmic RAD23B result?
Assess nuclear and cytoplasmic compartments separately: tissue IHC reports both across tissues, while cell imaging supports nucleoplasmic and cytosolic localisation (HPA: tissue IHC profile; HPA: subcellular). RAD23B distribution also changes with the cell cycle: nuclear levels decrease during S phase, and mitotic protein relocates to cytoplasm without chromatin association (UniProt P54727: subcellular location). Compare cells with similar morphology and proliferation context before calling a cytoplasmic pattern aberrant (UniProt P54727: subcellular location; standard IHC practice). Retain a nuclear counterstain and examine intact cell borders so diffuse chromogen is not assigned to the wrong compartment (standard IHC practice). A uniform stromal haze warrants background controls before biological interpretation (standard IHC practice).
Can isoforms or epitope accessibility change the RAD23B pattern?
RAD23B has 2 reported isoforms and several mapped phosphorylated residues, including positions 155, 160 and 164 (UniProt P54727: isoforms and modified residues). The supplied antibody evidence does not map the catalog antibody’s epitope, so its isoform coverage and sensitivity to those modifications remain unknown (supplied product evidence). Check the antibody’s immunogen or epitope documentation before interpreting a compartment difference as isoform specific (standard IHC practice). Compare retrieval conditions and antibody concentration on adjacent sections, using the same detection run to limit technical variation (standard IHC practice). Report the observed staining pattern without assigning it to a particular isoform unless an isoform-specific reagent validates that assignment (standard IHC practice).
How can IF help assess a disputed RAD23B IHC pattern?
Use IF as a separate localisation check: cell imaging supports mainly nucleoplasmic RAD23B with additional cytosolic signal (HPA: subcellular). Multiplex RAD23B with an independently validated marker for the cell population being assessed, and inspect separate channels before interpreting overlap (standard IF practice). Choose a fluorophore channel with low tissue autofluorescence, then include single-channel and no-primary controls to identify bleed-through and background (standard IF practice). RAD23B has no transmembrane segment and occurs inside cells, so permeabilisation must permit access to its intracellular epitope; the catalog antibody’s precise epitope is unspecified (UniProt P54727: topology and localisation; supplied product evidence). Do not transfer the paraffin-section caption’s unstated fixation conditions to IF (caption A02174-2).
What should I check when RAD23B chromogenic staining looks diffuse?
Run a no-primary control to separate detection-system background from antibody-dependent staining, and inspect the peptide-preabsorbed control where available (standard IHC practice; caption A02174-2). The selected image reports a peptide-preabsorbed negative control, but its caption does not establish specificity in every tissue or processing condition (caption A02174-2). Block endogenous peroxidase before chromogenic detection and check whether pigment or precipitated chromogen persists in controls (standard IHC practice). If background rises with signal, compare antibody titrations around the caption’s 1:100 condition while keeping retrieval at Tris-EDTA pH 8.0 (caption A02174-2; datasheet A02174-2). Preserve morphology and counterstain to distinguish cellular signal from diffuse deposits (standard IHC practice).
How should I score RAD23B across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Score nuclear and cytoplasmic staining separately because both compartments are reported in tissue IHC and their balance can vary with cell cycle (HPA: tissue IHC profile; UniProt P54727: subcellular location). For each predefined cell population, record percentage positive and intensity, or calculate a compartment-specific H-score using a fixed scoring rubric (standard IHC practice). If the question concerns infiltrating cells, report positive-cell density per mm² of viable tissue and the total density of that cell population (standard IHC practice). Normalise comparisons to the same cell type, viable tissue area, staining batch and exposure to detection reagents (standard IHC practice). Keep scoring thresholds fixed using appropriate controls (standard IHC practice).
When is a RAD23B-positive area likely to be artefactual?
A credible result should resolve to identifiable cells and plausible nuclear or cytoplasmic compartments; both are supported for RAD23B (HPA: tissue IHC profile; UniProt P54727: subcellular location). Treat signal confined to section edges, folds or necrotic areas cautiously, and compare those regions with intact tissue on the same slide (standard IHC practice). Check unexpected staining in the wrong cell population against morphology, an appropriate cell marker and control sections before assigning it to RAD23B (standard IHC practice). If brown signal remains in the no-primary control, investigate endogenous enzyme activity or detection background before scoring it (standard IHC practice). Peptide preabsorption supports the selected image’s staining assessment but does not independently prove every cellular assignment (caption A02174-2).
Boster reagents
Best RAD23B / Lysine-specific demethylase RAD23B IHC Antibodies
The catalog antibodies have IHC images from human paraffin sections (catalog image captions). M02174 also has a HeLa ICC image; listed reactivity covers human, mouse, rat, and monkey (catalog).
Real IHC data
Anti-Rad23B Antibody
Cat # A02174-2
Real IHC data
Anti-Rad23B (5H1) monoclonal Antibody
Cat # M02174
A02174-2 will render with human brain paraffin-section IHC at 1:100 after Tris-EDTA retrieval at pH 8.0 (A02174-2 image caption). M02174 will render with human prostate cancer paraffin-section IHC using citrate retrieval at pH 6.0; its separate image shows HeLa ICC (M02174 image captions).
Which to pick: For tissue IHC, A02174-2 is a rabbit polyclonal option illustrated in human brain, while M02174 is a mouse monoclonal option illustrated in prostate cancer; both captions specify paraffin sections, and neither reports the fixative (catalog; respective IHC image captions). For cell staining, M02174 has a HeLa ICC image, while A02174-2 lists IF with a dilution range of 1:200–1:1000 but has no IF image in this payload (catalog). For listed species coverage, choose M02174 when monkey reactivity is needed; both list human, mouse, and rat reactivity (catalog).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P54727 (RD23B_HUMAN, Lysine-specific demethylase RAD23B).
- Human Protein Atlas. RAD23B tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. RAD23B subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the cytosol..
- Human Protein Atlas. RAD23B antibody validation summary (3 antibodies).
- RAD23B Promotes Colorectal Cancer Metastasis via the Talin1/Integrin/PI3K/AKT/MMP9 Axis. Oncology research 2025 — PMC12573188.
- Rad23b exacerbates pathological aggregates through disrupting proteasome functions in Spinocerebellar ataxia type 3. Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics 2026 — PMC13224077.
- Differential Gene Expression in Human Hippocampus With Aging. Aging cell 2026 — PMC13140639.
- miR-196a reduces mutant Huntingtin aggregates by Rad23b-mediated degradation in Huntington's disease. Journal of biomedical science 2026 — PMC13613857.
- PubMed PMID:8168482 — UniProt-cited evidence.
- PubMed PMID:15064313 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RAD23B (Lysine-specific demethylase RAD23B). Expected localisation Nuclear and cytoplasmic staining across tissues (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 8.0 HIER, heat-mediated (datasheet A02174-2). Validated IHC antibody: A02174-2 at 1:100-1:300 (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 brain tissue; fixative not specified (datasheet A02174-2); 2. Fixation: Image fixative and duration unreported (datasheet A02174-2); 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: Tris-EDTA pH 8.0 (datasheet A02174-2); 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-RAD23B, 1:100-1:300 (datasheet A02174-2); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Cell cycle can shift the nuclear/cytoplasmic balance (UniProt); Isoform / epitope: 2 isoforms; epitope differences are unspecified (UniProt); Regulation: Nuclear levels decrease in S phase (UniProt). Evidence: UniProt P54727, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A02174-2, PMC12573188, PMC13140639. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RAD23B/immunohistochemistry-ihc-guide.