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RUNX3 / Runt-related transcription factor 3 · IHC design guide
Design Immunohistochemistry for RUNX3
Plan chromogenic RUNX3 IHC in paraffin sections using the catalog antibody at 0.5–1 μg/mL (datasheet PB9432). Assess nuclear staining in a subset of immune cells (HPA tissue IHC), while considering TGF-β-dependent nuclear translocation when interpreting localisation (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RUNX3 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 a subset of immune cells (HPA tissue IHC) | |
| Staining pattern | A subset of immune cells shows nuclear staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PB9432) | |
| Positive control | Skin+2 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 paraffin sections (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image PB9432) | |
| Caveat | Antibody–RNA staining consistency is medium (HPA tissue IHC) | |
| Regulation | TGF-β promotes nuclear translocation (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage is unknown (UniProt) |
Section 1
Recommended RUNX3 IHC & IF Protocols
The catalog antibody has an IHC protocol (datasheet: PB9432); the three published protocols below provide additional examples for RUNX3 staining (PMC5652736; PMC2427338; PMC3572981).
| Sample | Paraffin-embedded mouse spleen tissue; fixative not specified (datasheet PB9432) |
| Fixation | Image fixative and duration unreported (datasheet PB9432); 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 PB9432); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9432) |
| Primary antibody | Rabbit anti-RUNX3, 0.5-1μg/ml (datasheet PB9432) |
| Primary incubation | Overnight at 4 °C (datasheet PB9432) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9432) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | RUNX3-positive staining in langerhans cells of skin (HPA tissue IHC: Medium). HPA tissue profile: Nuclear expression in a subset of immune cells. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA at pH 8.0 (datasheet: PB9432); citrate at pH 6.0 is a published alternative (PMC5652736 methods).
Section 2
What Is the Expected RUNX3 Staining Pattern?
RUNX3 is a non-membrane transcription factor localized to the nucleus and cytoplasm; tyrosine-phosphorylated RUNX3 can localize to the cytoplasm (UniProt Q13761 localization and topology). In paraffin-section IHC, expect chiefly nuclear staining in a subset of immune cells (HPA tissue IHC). HPA rates the tissue profile Enhanced, while reporting only medium consistency between antibody staining and RNA expression (HPA tissue IHC).
| Discrete nuclear staining in some immune cells, especially tonsil non-germinal center cells or skin Langerhans cells (HPA tissue IHC). | This fits the reported tissue profile; both named populations show Medium staining (HPA tissue IHC). Assess individual cells and compartments instead of treating an entire section as uniformly positive. A compatible pattern supports interpretation, but HPA’s medium staining–RNA consistency limits how confidently staining alone identifies RUNX3 (HPA tissue IHC). |
| Predominantly cytoplasmic staining in otherwise plausible positive cells (UniProt Q13761 localization). | Review nuclear staining before calling the slide positive: HPA’s tissue profile is nuclear in a subset of immune cells (HPA tissue IHC). Cytoplasmic signal is biologically possible because phosphorylated RUNX3 can localize there, and TGF-beta stimulation can drive nuclear translocation (UniProt Q13761 localization). Cytoplasm alone therefore does not establish either a true positive or an artifact. |
| Broad staining in cells reported as negative, such as adipocytes or bronchial respiratory epithelium (HPA tissue IHC). | That distribution conflicts with those HPA observations and raises possible antibody cross-reactivity or endogenous detection activity (HPA tissue IHC; general IHC practice). Inspect a no-primary control and compare with a known-positive compartment before assigning the signal to RUNX3. A negative HPA entry is a reference observation, not a guarantee for every specimen. |
| Diffuse color over nuclei, cytoplasm, and surrounding tissue without a clear cellular pattern (general IHC practice). | Nonspecific background can obscure the expected nuclear subset (HPA tissue IHC; general IHC practice). Check blocking, washes, detection conditions, and the no-primary control (general IHC practice). Do not score widespread haze as RUNX3 merely because some nuclei lie within it. |
| No staining in tonsil non-germinal center cells or skin Langerhans cells (HPA tissue IHC). | Those populations have Medium HPA staining and can serve as reference-positive material when present (HPA tissue IHC). If they remain blank, first review the tissue, antibody dilution, retrieval, and detection run (general IHC practice). A blank reference reduces confidence in interpreting a test section as RUNX3-negative. |
💡Expected RUNX3 appearanceCall a result pattern-compatible when a subset of immune-cell nuclei stains distinctly, with Medium examples in tonsil non-germinal center cells and skin Langerhans cells; widespread signal in HPA-negative cell populations or diffuse tissue haze warrants investigation (HPA tissue IHC; general IHC practice).
| Cell population and tissue | HPA reports Medium staining in skin Langerhans cells and tonsil non-germinal center cells, Low staining in several other immune-rich tissues, and no detection in listed negative populations (HPA tissue IHC). Select and score the relevant cells, not the tissue name alone. |
| Subcellular state | Nucleus and cytoplasm are both reported for RUNX3; tyrosine-phosphorylated RUNX3 localizes to cytoplasm, with nuclear translocation after TGF-beta stimulation (UniProt Q13761 localization). Interpret cytoplasmic signal in context of the predominantly nuclear tissue-IHC profile (HPA tissue IHC). |
| Antibody validation | HPA reports Enhanced IHC validation for HPA059006 and CAB025416, while HPA004195 has Supported ICC validation without an IHC status in the supplied list (HPA antibodies). Validation for one application does not establish performance in another (HPA antibodies; general IHC practice). |
| Isoforms and processing | UniProt lists two RUNX3 isoforms, a single 1–415 chain, no signal peptide or propeptide, and no transmembrane segment (UniProt Q13761). These facts support an intracellular interpretation; they do not identify which isoform a particular antibody recognizes. |
| IF/ICC Q&A | Where should IF signal appear? HPA reports mainly nucleoplasm and vesicles, with additional cytosol, in its ICC-IF summary (HPA subcellular). This is a compartment reference for the separate IF/ICC guide, not an IHC-P staining pattern or protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive reference cells are blank (HPA tissue IHC). | The run may have inadequate detection or unsuitable antibody conditions (general IHC practice); absence alone cannot identify which step failed. | Confirm that the expected cells are present, then check the antibody’s IHC-P instructions, retrieval, dilution, detection reagents, and run controls (general IHC practice). Reassess test negatives only after the reference stains. |
| Nearly every nucleus is colored, including HPA-negative populations (HPA tissue IHC). | The distribution is broader than HPA’s nuclear subset profile; nonspecific binding or detection background is possible (HPA tissue IHC; general IHC practice). | Compare matched positive and negative populations, inspect a no-primary control, and review blocking and antibody concentration (general IHC practice). Score cell-specific signal only after broad background is resolved. |
| Only diffuse cytoplasmic color is visible (UniProt Q13761 localization). | Cytoplasmic RUNX3 is possible, but this pattern does not reproduce the main nuclear tissue profile; background may also contribute (UniProt Q13761 localization; HPA tissue IHC; general IHC practice). | Inspect nuclear signal in reference-positive immune cells and compare with the no-primary control (HPA tissue IHC; general IHC practice). Record compartment separately from intensity rather than calling the slide definitively positive. |
| Brown precipitate or color persists in the no-primary control (general IHC practice). | Primary-independent detection activity or reagent background is possible (general IHC practice). | Review the detection system’s endogenous-activity blocking and wash steps, then repeat the control under the same detection conditions (general IHC practice). Do not attribute control-positive color to RUNX3. |
| A reported negative population stains while reference-positive cells stain appropriately (HPA tissue IHC). | Possible cross-reactivity or specimen-dependent expression needs resolution; HPA observations alone cannot prove either explanation (HPA tissue IHC; general IHC practice). | Document the exact cell population and compartment, compare with HPA’s reported pattern, and seek independent validation before making a biological claim (HPA tissue IHC; general IHC practice). |
| An IF/ICC image appears more vesicular than the paraffin IHC section (HPA subcellular; HPA tissue IHC). | HPA’s ICC-IF summary includes vesicles and cytosol, whereas its tissue-IHC profile emphasizes nuclear staining in a subset of immune cells (HPA subcellular; HPA tissue IHC). | Interpret each application against its own HPA reference and antibody validation status (HPA antibodies; HPA subcellular; HPA tissue IHC). Do not use an ICC-only validation status as evidence of IHC-P performance. |
Experiment evidence
IHC & IF images showing RUNX3 staining
PB9432 · IHCIHC analysis of RUNX3 using anti-RUNX3 antibody (PB9432). RUNX3 was detected in a paraffin-embedded section of mouse spleen tissue. Heat med
PB9432 · IHCIHC analysis of RUNX3 using anti-RUNX3 antibody (PB9432). RUNX3 was detected in a paraffin-embedded section of rat spleen tissue. Heat media
PB9432 · IFIF analysis of RUNX3 using anti-RUNX3 antibody (PB9432). RUNX3 was detected in immunocytochemical section of U20S cells. Enzyme antigen retr
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000020633-RUNX3/tissue/cerebral+cortex#imid_16361259
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000020633-RUNX3/tissue/colon#imid_16357377
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000020633-RUNX3/tissue/liver#imid_16357386
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000020633-RUNX3/tissue/kidney#imid_16357371
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA004195; green = RUNX3, blue = nucleus, red = microtubules.
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA004195; green = RUNX3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RUNX3 IHC & IF
🧪Run skin first and look for RUNX3 staining in Langerhans cells (Medium; HPA: skin). Use adipose tissue as the negative comparator, where adipocytes are not detected (HPA: adipose tissue); on the skin slide, compare Langerhans cells with neighboring non-Langerhans cells, treating the latter as background comparators rather than confirmed RUNX3-negative cells (HPA: skin reports Langerhans cells only).
Positive control tissue: Skin (Langerhans cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RUNX3 in A-549, SiHa, U2OS, NIH 3T3, with annotated localisation: Nucleoplasm (supported), Vesicles (approved) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and concentration-matched rabbit isotype controls, matched to the catalog antibody’s clonality, plus RUNX3-knockout material if available as a biological negative (hero caption: rabbit antibody; standard IHC practice). For the caption’s biotin-based DAB detection, block endogenous peroxidase and biotin and distinguish brown staining from skin pigment (hero caption: biotinylated secondary, SABC and DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected PB9432 paraffin-section caption does not state the fixative (hero caption). The caption uses heat retrieval in EDTA, pH 8.0 on mouse spleen; retrieval conditions for skin need validation (hero caption). Paraffin-section IHC has a supplied example, while whether frozen sections or IF are easier is unreported; skin pigment can complicate chromogenic scoring (hero caption; standard IHC practice).
HPA tissue IHC evidence for RUNX3
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 |
|---|---|---|---|---|
| Skin | Langerhans cells | Medium | Protein (IHC) | HPA → |
| Small intestine | Enterocytes - Microvilli | Medium | Protein (IHC) | HPA → |
| Tonsil | Non-germinal center cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RUNX3 IHC Tips
Troubleshoot RUNX3 staining by checking retrieval, cell identity and nuclear localisation alongside the selected antibody’s documented paraffin section conditions.
How should I optimize retrieval when RUNX3 staining is weak in paraffin sections?
Start with heat-mediated antigen retrieval in EDTA buffer at pH 8.0 (datasheet PB9432). The selected paraffin section example used this retrieval before overnight incubation at 4°C with 1 μg/ml primary antibody (caption PB9432). Compare retrieval durations on adjacent sections while keeping antibody concentration, detection and development time constant (standard IHC practice). If staining remains weak, test a second retrieval condition only as a documented fallback, checking whether background or tissue damage increases (standard IHC practice). Judge improvement in the relevant cells against a no-primary control, rather than by overall DAB darkness (standard IHC practice).
Could fixation explain weak or uneven RUNX3 staining?
RUNX3-specific fixation sensitivity is unknown here: the selected paraffin section caption does not state its fixative (caption PB9432). Record the actual fixative, fixation interval, section age and processing history before changing retrieval or antibody concentration (standard IHC practice). For a new series, process comparison specimens consistently and assess adjacent sections under the same EDTA pH 8.0 retrieval condition (datasheet PB9432; standard IHC practice). Compare cell-level nuclear staining and tissue preservation, since harsh processing can complicate interpretation of a nuclear transcription factor (UniProt Q13761 localisation; standard IHC practice). Do not assign a RUNX3-specific fixation effect without a direct comparison (caption PB9432).
Should RUNX3 staining be nuclear, cytoplasmic or both?
Score nuclear staining explicitly: tissue IHC reports expression in a subset of immune cells, while subcellular imaging supports nucleoplasmic localisation (HPA tissue IHC; HPA subcellular). Cytoplasmic signal is biologically possible because RUNX3 also localises to the cytoplasm, and its tyrosine-phosphorylated form is reported there (UniProt Q13761 localisation). RUNX3 can move from cytoplasm to nucleus after TGF-beta stimulation, so record compartments separately instead of collapsing them into one positive score (UniProt Q13761 localisation). Interpret vesicular or diffuse cytoplasmic DAB cautiously, comparing morphology, controls and nuclear staining in the same section (HPA subcellular; standard IHC practice). Document the fraction of positive cells in each compartment (standard IHC practice).
Can isoforms or epitope accessibility change the RUNX3 IHC pattern?
RUNX3 has 2 listed isoforms and a Runt domain spanning residues 54–182 (UniProt Q13761 isoforms and domains). The supplied product caption does not identify the antibody epitope, so recognition of both isoforms or a particular modified form remains unestablished (caption PB9432). A phosphoserine is annotated at residue 243, but that annotation alone does not show that fixation or retrieval alters this antibody’s binding (UniProt Q13761 modified residues). If isoform discrimination matters, obtain the immunogen or epitope information and compare it with each isoform sequence before interpreting staining differences (standard antibody validation practice). Keep retrieval and detection identical across comparison sections (standard IHC practice).
How can I check a RUNX3 IHC finding by multiplex IF?
Treat multiplex IF as a separate validation experiment because the supplied antibody example documents chromogenic staining of a paraffin section, with no IF conditions supplied (caption PB9432). Pair RUNX3 with a validated marker for the expected immune-cell population and inspect nuclear overlap at single-cell resolution (HPA tissue IHC; standard IF practice). Choose a far-red fluorophore if the specimen has substantial shorter-wavelength autofluorescence, and include unstained and single-channel controls (standard IF practice). Because RUNX3 has no transmembrane segment and a nuclear localisation, test permeabilisation sufficient for nuclear access while checking morphology (UniProt Q13761 topology and localisation; standard IF practice). Epitope accessibility remains uncertain until the antibody’s epitope is established (caption PB9432).
What should I check when DAB background obscures RUNX3-positive cells?
The selected example used 10% goat serum blocking, a biotinylated secondary antibody, an avidin-biotin detection complex and DAB (caption PB9432). Run no-primary and detection-only sections to assess secondary binding, endogenous enzyme signal and detection background (standard IHC practice). For peroxidase-based DAB detection, verify the peroxidase block and consider endogenous biotin when using an avidin-biotin system (standard IHC practice). Titrate the primary around the documented 1 μg/ml condition and shorten development only while monitoring the expected cell-level nuclear signal (caption PB9432; HPA tissue IHC; standard IHC practice). Compare section edges with the interior before calling diffuse brown staining specific (standard IHC practice).
How should I quantify heterogeneous RUNX3 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because tissue IHC reports nuclear expression in a subset of immune cells (HPA tissue IHC). For chromogenic sections, report the percentage of positive eligible cells and, when intensity is reproducible, a nuclear H-score from 0–300 (standard IHC scoring practice). If the question concerns infiltration, also report positive-cell density per mm² of viable tissue (standard IHC scoring practice). Normalize counts to the number of eligible cells or measured viable area, and use the same staining threshold across the series (standard IHC scoring practice). Record cytoplasmic staining separately from nuclear staining (UniProt Q13761 localisation; standard IHC scoring practice).
How can I distinguish true RUNX3 staining from tissue artefact?
A convincing pattern identifies discrete cells and a plausible compartment: HPA tissue IHC reports nuclear staining in a subset of immune cells (HPA tissue IHC). Cytoplasmic staining needs careful assessment rather than automatic rejection, because RUNX3 can localise there, particularly in its tyrosine-phosphorylated form (UniProt Q13761 localisation). Be cautious with signal confined to section edges, necrotic regions or damaged cells, and compare it with intact interior tissue (standard IHC practice). Check no-primary and detection controls for endogenous peroxidase or avidin-biotin background before attributing DAB to RUNX3 (standard IHC practice). Confirm that the scored cells match the intended cell population on the counterstained section (standard IHC practice).
Boster reagents
Best RUNX3 / Runt-related transcription factor 3 IHC Antibodies
The IHC-validated antibody PB9432 has paraffin-section images from mouse and rat spleen and human intestinal and lung cancer tissue (PB9432 IHC captions), plus an IF image from U20S cells (PB9432 IF caption).
Real IHC data
Anti-RUNX3 Antibody ®
Cat # PB9432
PB9432 has IHC images from paraffin sections of mouse and rat spleen and human intestinal and lung cancer tissue (PB9432 IHC captions). It is also listed for IF/ICC and has an IF image from U20S cells (PB9432 applications; PB9432 IF caption).
Which to pick: For tissue IHC, choose PB9432: its images show paraffin sections, while the fixative is unreported (PB9432 IHC captions). For IF/ICC, PB9432 is listed for both applications and has an IF image from U20S cells (PB9432 applications; PB9432 IF caption). For work across human, mouse and rat samples, PB9432 lists reactivity with all three and has IHC images from each species; it has a rabbit host, but clonality is unreported (PB9432 reactivity; PB9432 IHC captions; PB9432 catalog host and clone fields).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q13761 (RUNX3_HUMAN, Runt-related transcription factor 3).
- Human Protein Atlas. RUNX3 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. RUNX3 subcellular location (ICC-IF): Mainly localized to the nucleoplasm and vesicles. In addition localized to the cytosol..
- Human Protein Atlas. RUNX3 antibody validation summary (3 antibodies).
- H. pylori-Eradication Therapy Increases RUNX3 Expression in the Glandular Epithelial Cells in Enlarged-Fold Gastritis. Journal of clinical biochemistry and nutrition 2010 — PMC2872232.
- Combination immunohistochemistry for SMAD4 and Runt-related transcription factor 3 may identify a favorable prognostic subgroup of pancreatic ductal adenocarcinomas. Oncotarget 2017 — PMC5652736.
- Enhancer of zeste homologue 2 (EZH2) down-regulates RUNX3 by increasing histone H3 methylation. The Journal of biological chemistry 2008 — PMC2427338.
- RUNX3 suppresses migration, invasion and angiogenesis of human renal cell carcinoma. PloS one 2013 — PMC3572981.
- PubMed PMID:7622058 — UniProt-cited evidence.
- PubMed PMID:7835892 — UniProt-cited evidence.
- PubMed PMID:16710414 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RUNX3 (Runt-related transcription factor 3). Expected localisation Nuclear staining in a subset of immune cells (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PB9432). Validated IHC antibody: PB9432 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Skin (Langerhans cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded mouse spleen tissue; fixative not specified (datasheet PB9432); 2. Fixation: Image fixative and duration unreported (datasheet PB9432); 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 PB9432); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9432); 8. Primary antibody: Rabbit anti-RUNX3, 0.5-1μg/ml (datasheet PB9432); 9. Primary incubation: Overnight at 4 °C (datasheet PB9432). Watch for: Fixation: Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image PB9432); Caveat: Antibody–RNA staining consistency is medium (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage is unknown (UniProt); Regulation: TGF-β promotes nuclear translocation (UniProt). Evidence: UniProt Q13761, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm and vesicles. In addition…, Datasheet PB9432, PMC5652736, PMC2427338. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RUNX3/immunohistochemistry-ihc-guide.