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KDM5B / Lysine-specific demethylase 5B · IHC design guide
Design Immunohistochemistry for KDM5B
Plan KDM5B IHC-P around nuclear staining, using testis pachytene spermatocytes as a strong positive reference (HPA tissue IHC). The guide covers the catalog antibody's 0.5–1 μg/mL dilution range and chromogenic detection (datasheet PB10073).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
KDM5B 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 staining in tissue sections (HPA tissue IHC) | |
| Staining pattern | Widespread nuclear staining, strongest in testis (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PB10073) | |
| Positive control | Testis+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Signal varies by cell type and may be weak outside testis (HPA tissue IHC) | |
| Regulation | Highest in testis; up-regulated in breast cancer (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage is unspecified (UniProt; datasheet PB10073) |
Section 1
Recommended KDM5B IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: PB10073) is accompanied by published KDM5B IHC methods (PMC2848192; PMC6210936; PMC10154691).
| Sample | Paraffin-embedded human intestinal cancer tissue; fixative not specified (datasheet PB10073) |
| Fixation | Image fixative and duration unreported (datasheet PB10073); 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 PB10073); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB10073) |
| Primary antibody | Rabbit anti-KDM5B, 0.5-1μg/ml (datasheet PB10073) |
| Primary incubation | Overnight at 4 °C (datasheet PB10073) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB10073) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | KDM5B-positive staining in pachytene spermatocytes of testis (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear expression, most abundant in testis. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval (datasheet: PB10073). Microwave citrate pH 6.0 is a published alternative (PMC6210936).
Section 2
What Is the Expected KDM5B Staining Pattern?
KDM5B is a nuclear protein without a transmembrane segment (UniProt Q9UGL1: subcellular location and topology). In paraffin-section IHC, expect chiefly nuclear staining across tissues, strongest in testicular pachytene spermatocytes (HPA: ubiquitous nuclear expression; High in pachytene spermatocytes). HPA rates its tissue IHC profile Enhanced, citing high consistency between antibody staining and RNA expression (HPA: tissue IHC reliability).
| Strong nuclear stain in pachytene spermatocytes; weaker nuclear stain in some other cells. | This fits the testis-rich pattern: pachytene spermatocytes are High, while several listed respiratory and reproductive cell types are Medium (HPA: tissue IHC). Compare the named cell populations when judging intensity. |
| Staining is predominantly cytoplasmic or appears outside cells, with little nuclear signal. | For IHC, treat this as a possible localization artefact and inspect the negative control and detection steps (UniProt Q9UGL1: nucleus; HPA: ubiquitous nuclear IHC). HPA reports uncertain additional cytosolic localization in ICC-IF, so a minor cytosolic IF signal alone is inconclusive (HPA: subcellular). |
| Prominent stain appears in adipocytes or skeletal-muscle myocytes. | These cell populations are listed as Not detected; conspicuous staining merits a specificity check, including cross-reactivity or endogenous detection activity (HPA: tissue IHC; general IHC practice). “Not detected” describes the reported staining result, not proof that every specimen must be blank. |
| Diffuse color obscures nuclei and tissue boundaries throughout the section. | This cannot establish KDM5B localization. Compare a no-primary control and review blocking, washes, detection and counterstain before scoring (general IHC practice). The expected reference pattern is nuclear, with the strongest listed staining in pachytene spermatocytes (HPA: tissue IHC). |
| No nuclear signal is visible in a testis section containing pachytene spermatocytes. | The expected High cell population is missing its reference signal (HPA: tissue IHC). Check that those cells are present, then review antibody validation, the retrieval and detection controls, and the run record (general IHC practice). Do not infer target-specific fixation sensitivity from this result. |
💡Expected KDM5B appearanceA convincing positive is chiefly nuclear, reaching High intensity in testicular pachytene spermatocytes (HPA: tissue IHC); widespread staining outside nuclei, especially in listed Not detected cell populations, warrants investigation.
| Reference tissue and cell population | Testis is the strongest listed comparator, specifically pachytene spermatocytes at High; bronchial ciliated cells and epididymal glandular cells are Medium (HPA: tissue IHC). Score the named cells rather than assigning one intensity to an entire section. |
| Antibody-specific IHC evidence | HPA027179 and CAB068193 have Enhanced IHC status; HPA053723 has no listed IHC status in this payload (HPA: antibody validation). Apply validation to the antibody used, rather than transferring another antibody's status. |
| Nuclear localization | UniProt assigns KDM5B to the nucleus, and HPA describes ubiquitous nuclear tissue IHC (UniProt Q9UGL1: subcellular location; HPA: tissue IHC). Judge positive cells by nuclear staining; a general chromogenic haze does not establish localization. |
| Isoforms and antibody recognition | UniProt lists 2 isoforms (UniProt Q9UGL1: isoforms). The supplied record gives no antibody epitope or isoform coverage, so it cannot predict which isoform an IHC stain detects; consult the antibody's documented validation before interpreting discordant results. |
| IF/ICC question: should its signal match IHC exactly? | Expect mainly nucleoplasmic IF signal; HPA marks additional cytosolic IF localization uncertain (HPA: subcellular). Treat that as an IF interpretation caveat, not an IHC protocol choice or evidence that diffuse cytoplasmic chromogen is specific. |
| Situation | Likely cause | Next action |
|---|---|---|
| Nuclei are pale, but cytoplasm carries most of the chromogen. | The distribution conflicts with the nuclear IHC profile (HPA: tissue IHC; UniProt Q9UGL1: nucleus); nonspecific detection or poor localization is possible (general IHC practice). | Compare positive and no-primary controls, inspect morphology, and review detection and counterstain. Score as unresolved until nuclear localization is credible (general IHC practice). |
| Adipocytes, myocytes or germinal-center cells stain prominently. | These named populations were Not detected in the reported tissue IHC; cross-reactivity or endogenous detection activity could explain unexpected color (HPA: tissue IHC; general IHC practice). | Check a no-primary control and relevant detection controls, then confirm the antibody's own IHC validation before calling those cells positive (general IHC practice). |
| Color covers tissue and background, masking nuclear boundaries. | Diffuse background prevents compartment scoring and may arise from blocking, washing or detection conditions (general IHC practice). | Inspect a no-primary control; review blocking, washes, chromogen development and counterstain, then repeat scoring only where nuclei remain distinguishable (general IHC practice). |
| Pachytene spermatocytes are present but show no nuclear stain. | This conflicts with their reported High staining (HPA: tissue IHC). The result alone does not identify a KDM5B-specific fixation or retrieval effect. | Verify cell identification and the antibody's IHC evidence; review the run's retrieval, reagent and detection controls as general IHC checks (general IHC practice). |
| A weak section is judged negative because nearby cells stain more strongly. | Reported intensity varies by cell population: skin granular-layer cells are Low, while pachytene spermatocytes are High (HPA: tissue IHC). | Identify and score the relevant cells and nuclear compartment first. Use the reported cell-specific levels as context, without treating a Low result as proof of assay failure (HPA: tissue IHC). |
| IF shows some cytosolic signal although IHC appears nuclear. | HPA supports nucleoplasmic ICC-IF localization and labels additional cytosolic localization uncertain (HPA: subcellular); the IHC profile is nuclear (HPA: tissue IHC). | Interpret each application against its own reported pattern. Check IF controls and nuclear counterstain, and avoid using the uncertain IF signal to validate cytoplasmic IHC color (general IHC practice; HPA: subcellular). |
Experiment evidence
IHC & IF images showing KDM5B staining
PB10073 · IHCIHC analysis of KDM5B using anti-KDM5B antibody (PB10073). KDM5B was detected in paraffin-embedded section of human intestinal cancer tissue
PB10073 · IHCIHC analysis of KDM5B using anti-KDM5B antibody (PB10073). KDM5B was detected in paraffin-embedded section of human intestinal cancer tissue
PB10073 · IFIF analysis of KDM5B using anti-KDM5B antibody (PB10073). KDM5B was detected in immunocytochemical section of MCF-7 cells. Enzyme antigen re
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000117139-KDM5B/tissue/cerebral+cortex#imid_18803538
HPA · EsophagusHuman Protein Atlas IHC, Esophagus. Source: https://www.proteinatlas.org/ENSG00000117139-KDM5B/tissue/esophagus#imid_18803518
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000117139-KDM5B/tissue/colon#imid_18800640
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000117139-KDM5B/tissue/liver#imid_18800633
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA053723; green = KDM5B, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA053723; green = KDM5B, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for KDM5B IHC & IF
🧪Run testis first and look for nuclear staining in pachytene spermatocytes (HPA: High in pachytene spermatocytes; UniProt Q9UGL1: nucleus). Use adipose tissue as the negative tissue (HPA: Not detected in adipocytes); the supplied testis data do not identify a validated internal-negative cell type, so neighboring cells without nuclear signal can serve only as background comparators (HPA: testis row).
Positive control tissue: Testis (Pachytene spermatocytes, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show KDM5B in A-431, U-251MG, U2OS, RPTEC/TERT1, hTERT-RPE1 (serum starved), KOLF2.1J, NIH 3T3, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide and a matched rabbit control IgG; assess specificity with KDM5B-knockout material or peptide competition if the cognate peptide is available (PB10073 tissue-IHC caption: rabbit primary; standard IHC controls). For testis sections, quench endogenous peroxidase and check for endogenous biotin background when using the documented biotinylated secondary and SABC-DAB detection (PB10073 tissue-IHC caption: detection system; standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected PB10073 paraffin-section caption does not state the fixative (PB10073 tissue-IHC caption: fixative unreported). Heat retrieval in EDTA at pH 8.0 is documented for the caption’s intestinal cancer section and needs validation in testis (PB10073 tissue-IHC caption: retrieval and tissue). The evidence does not establish whether frozen sections or IF would be easier; in testis, score pachytene spermatocyte nuclei by cell type to avoid confusing staining in neighboring cells with the expected positive population (HPA: High in pachytene spermatocytes; HPA subcellular: nucleoplasm supported).
HPA tissue IHC evidence for KDM5B
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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 |
|---|---|---|---|---|
| Testis | Pachytene spermatocytes | High | Protein (IHC) | HPA → |
| Bronchus | Ciliated cells (cell body) | Medium | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | Medium | Protein (IHC) | HPA → |
| Fallopian tube | Ciliated cells (cell body) | Medium | Protein (IHC) | HPA → |
| Lung | Alveolar cells type I | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cerebral cortex | Glial cells | Not detected | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced KDM5B IHC Tips
Troubleshoot KDM5B staining by checking nuclear localisation, retrieval conditions, controls and cell-specific scoring before interpreting chromogenic signal.
What retrieval should I use when nuclear KDM5B staining is weak?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet PB10073). The catalog antibody stained human intestinal cancer tissue after this retrieval, with 1 μg/ml primary antibody incubated overnight at 4°C (datasheet PB10073). If nuclear staining remains weak, check that sections were fully deparaffinised, that the retrieval solution reached its intended temperature, and that the run included a positive control; these are general IHC checks (standard IHC practice). Compare any adjusted heating conditions on matched sections, because an apparent gain in signal can also increase tissue damage or diffuse chromogenic background (standard IHC practice).
Could fixation explain variable KDM5B staining between paraffin blocks?
Target-specific fixation sensitivity for KDM5B is unknown from the supplied evidence: the selected paraffin-section caption does not state a fixative (datasheet PB10073). Record each block's fixative and fixation duration, then compare sections processed with the same EDTA pH 8.0 retrieval and detection run (datasheet PB10073; standard IHC practice). Include a concurrently stained positive-control section and examine morphology before attributing a weak nucleus to low KDM5B expression (standard IHC practice). Neither the nuclear localisation nor the reported acetylation and phosphorylation sites establishes a particular fixation requirement or explains a block-to-block difference (UniProt Q9UGL1; standard IHC practice).
How should I assess cytoplasmic staining when KDM5B is expected in nuclei?
Prioritise nuclear chromogenic signal when assessing KDM5B: UniProt places the protein in the nucleus, and the tissue IHC profile describes ubiquitous nuclear expression (UniProt Q9UGL1; HPA tissue IHC). HPA subcellular data support nucleoplasmic localisation and list additional cytosolic localisation as uncertain, so isolated cytoplasmic DAB deserves separate scrutiny (HPA subcellular). Inspect a hematoxylin counterstain to distinguish nuclei from cytoplasm, then compare the suspect cells with a concurrently stained positive control and a no-primary control (standard IHC practice). Score nuclear and cytoplasmic staining separately, recording cell type and intensity rather than combining both compartments into one positive call (standard IHC practice).
Can alternative isoforms or epitope masking explain discordant IHC results?
KDM5B has 2 listed isoforms, but the supplied antibody caption does not identify its epitope or establish isoform recognition (UniProt Q9UGL1; datasheet PB10073). The protein contains JmjN, ARID and JmjC domains, and has reported acetylation and phosphorylation sites; those annotations alone do not show that this antibody is affected by a particular modification (UniProt Q9UGL1). When two IHC antibodies disagree, obtain their documented immunogen regions and compare staining in matched sections under each validated retrieval condition (standard IHC practice). Keep interpretation tied to cell type and nuclear localisation, and avoid assigning a discordant signal to an isoform without direct epitope or isoform-specific evidence (HPA tissue IHC; standard IHC practice).
How can IF help assess KDM5B localisation alongside chromogenic IHC?
For a separate IF assessment, multiplex KDM5B with a marker identifying the expected cell population, such as pachytene spermatocytes in testis, and use a nuclear counterstain to check overlap (HPA tissue IHC; standard IF practice). Choose a fluorophore channel with low tissue autofluorescence and include single-label controls so bleed-through does not mimic colocalisation (standard IF practice). KDM5B has no transmembrane segment and is chiefly nuclear, so permeabilisation must permit antibody access through the plasma membrane to the nuclear compartment (UniProt Q9UGL1; standard IF practice). Optimise that IF permeabilisation on separate specimens; the paraffin-section retrieval and 1 μg/ml incubation belong to the catalog antibody's IHC evidence (datasheet PB10073).
What causes diffuse or granular background in KDM5B chromogenic IHC?
First compare suspect staining with a no-primary control, because persistent DAB there points toward the detection system or tissue-associated background rather than demonstrated KDM5B binding (standard IHC practice). The catalog image used 10% goat serum blocking, a biotinylated secondary, a streptavidin–biotin complex and DAB; those details identify steps to inspect when reproducing that method (datasheet PB10073). Check endogenous peroxidase blocking and, for a biotin-based system, assess endogenous biotin interference with appropriate controls (standard IHC practice). Titrate the primary and secondary on matched sections, wash consistently, and judge whether background falls while expected nuclear staining remains visible (UniProt Q9UGL1; standard IHC practice).
How should I quantify KDM5B across tissue regions? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and analysis region before scoring, then count nuclear-positive cells rather than treating every brown pixel as KDM5B expression (UniProt Q9UGL1; standard IHC practice). Report the percentage of positive nuclei and, if intensity is reproducible, an H-score from 0–300 based on the fractions at each intensity (standard IHC practice). For spatial comparisons, positive nuclei per mm² can accompany the fraction of positive nuclei; normalise counts to evaluable cells or area and exclude necrotic or damaged regions by a preset rule (standard IHC practice). Keep retrieval, detection and scoring thresholds consistent across sections, with a shared control to monitor run-to-run variation (standard IHC practice).
When is a positive KDM5B IHC signal convincing?
A convincing call shows staining in intact nuclei within an identified cell population, consistent with KDM5B's nuclear annotation and the HPA tissue IHC pattern (UniProt Q9UGL1; HPA tissue IHC). Pachytene spermatocytes provide a strongly stained reference population, whereas HPA reports no detection in adipocytes; these observations guide control selection without making every cell in either tissue an absolute control (HPA tissue IHC). Discount staining confined to section edges, folds or necrotic regions, and check a no-primary control for detection-system or endogenous enzyme signal (standard IHC practice). Evaluate unexpected cytoplasmic staining separately because HPA supports nucleoplasmic localisation while describing the additional cytosolic location as uncertain (HPA subcellular).
Boster reagents
Best KDM5B / Lysine-specific demethylase 5B IHC Antibodies
PB10073 has IHC data from paraffin sections of human intestinal, lung and breast cancer tissue, plus IF data from MCF-7 cells (catalog image captions).
Real IHC data
Anti-KDM5B/PLU1/Jarid1B Antibody ®
Cat # PB10073
PB10073 is listed for IHC and IF/ICC, with stated reactivity to human, monkey, mouse and rat (catalog applications and reactivity). Its IHC captions show human intestinal, lung and breast cancer paraffin sections; its IF caption shows MCF-7 cells (catalog image captions).
Which to pick: Choose PB10073 for tissue IHC: its images document paraffin sections, with a starting range of 0.5–1 μg/ml (catalog IHC captions; catalog IHC dilution). The same rabbit antibody supports IF/ICC at 2 μg/ml, with an IF image from MCF-7 cells (catalog host, applications, dilution and IF caption). For cross-species planning, its stated reactivity includes human, monkey, mouse and rat, but the supplied IHC images show human tissue only; the fixative is unreported (catalog reactivity and IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9UGL1 (KDM5B_HUMAN, Lysine-specific demethylase 5B).
- Human Protein Atlas. KDM5B tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. KDM5B subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the cytosol..
- Human Protein Atlas. KDM5B antibody validation summary (3 antibodies).
- Aberrant KDM5B expression promotes aggressive breast cancer through MALAT1 overexpression and downregulation of hsa-miR-448. BMC cancer 2016 — PMC4768424.
- Overexpression of the JmjC histone demethylase KDM5B in human carcinogenesis: involvement in the proliferation of cancer cells through the E2F/RB pathway. Molecular cancer 2010 — PMC2848192.
- Increased Expression of Lysine-Specific Demethylase 5B (KDM5B) Promotes Tumor Cell Growth in Hep3B Cells and is an Independent Prognostic Factor in Patients with Hepatocellular Carcinoma. Medical science monitor : international medical journal of experimental and clinical research 2018 — PMC6210936.
- The KDM5B and KDM1A lysine demethylases cooperate in regulating androgen receptor expression and signalling in prostate cancer. Frontiers in cell and developmental biology 2023 — PMC10154691.
- PubMed PMID:10336460 — UniProt-cited evidence.
- PubMed PMID:10616211 — UniProt-cited evidence.
- PubMed PMID:10878660 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for KDM5B (Lysine-specific demethylase 5B). Expected localisation Nuclear staining in tissue sections (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PB10073). Validated IHC antibody: PB10073 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Pachytene spermatocytes); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human intestinal cancer tissue; fixative not specified (datasheet PB10073); 2. Fixation: Image fixative and duration unreported (datasheet PB10073); 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 PB10073); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB10073); 8. Primary antibody: Rabbit anti-KDM5B, 0.5-1μg/ml (datasheet PB10073); 9. Primary incubation: Overnight at 4 °C (datasheet PB10073). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Signal varies by cell type and may be weak outside testis (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage is unspecified (UniProt; datasheet PB10073); Regulation: Highest in testis; up-regulated in breast cancer (UniProt). Evidence: UniProt Q9UGL1, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet PB10073, PMC2848192, PMC6210936. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/KDM5B/immunohistochemistry-ihc-guide.