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RUNX1T1 / Protein CBFA2T1 · IHC design guide
Design Immunohistochemistry for RUNX1T1
Plan chromogenic paraffin IHC for RUNX1T1 using the catalog antibody at 0.5–1 μg/mL (datasheet A01792-1). Compare medium staining in testis Leydig cells with undetected staining in adrenal glandular cells, and interpret results cautiously because tissue staining has low consistency with RNA expression (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RUNX1T1 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 | Low nucleolar tissue signal (HPA tissue IHC); nuclear dots (UniProt) | |
| Staining pattern | Low nucleolar signal in most tissues; Leydig cells stain at medium intensity (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A01792-1) | |
| Positive control | Bronchus+3 more · see all | |
| Negative control | Adrenal gland+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation conditions consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Tissue staining has low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | Brain-enhanced RNA expression (HPA RNA) | |
| Isoform / epitope | 6 isoforms; check epitope coverage across variants (UniProt) |
Section 1
Recommended RUNX1T1 IHC & IF Protocols
The catalog antibody’s IHC-P protocol appears alongside published RUNX1T1 staining methods from two articles (PMC5143359; PMC11239676).
| Sample | Paraffin-embedded human mammary cancer tissue; fixative not specified (datasheet A01792-1) |
| Fixation | Image fixative and duration unreported (datasheet A01792-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet A01792-1) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A01792-1) |
| Primary antibody | Rabbit anti-RUNX1T1, 0.5-1μg/ml (datasheet A01792-1) |
| Primary incubation | Overnight at 4 °C (datasheet A01792-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A01792-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | RUNX1T1-positive staining in ciliated cells (ciliary rootlets) of bronchus (HPA tissue IHC: Medium). HPA tissue profile: Low nucleolar expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated citrate pH 6 retrieval (datasheet: A01792-1); the published gastric-section method specifies 10 mM citrate without a pH (PMC5143359).
Section 2
What Is the Expected RUNX1T1 Staining Pattern?
RUNX1T1 is a nuclear protein with no transmembrane segment (UniProt Q06455). In tissue IHC, expect generally low nucleolar staining, with medium staining reported in Leydig cells and ciliated-cell rootlets (HPA tissue IHC). Interpret these patterns cautiously: HPA rates the tissue IHC profile Approved but reports low consistency between antibody staining and RNA expression (HPA tissue IHC).
| Faint nucleolar staining across tissue, or medium staining in testis Leydig cells and bronchial, fallopian-tube, or nasopharyngeal ciliary rootlets (HPA tissue IHC). | These match the reported tissue IHC profile (HPA tissue IHC). Assess the named cell and subcellular structure before judging a field positive; UniProt also places RUNX1T1 in the nucleus and reports discrete nuclear dots with ATN1 (UniProt Q06455). |
| Broad cytoplasmic or membranous chromogen without a recognizable nucleolar or ciliary-rootlet pattern. | Treat this as suspicious for nonspecific staining or detection artefact (general IHC practice). RUNX1T1 has no transmembrane segment (UniProt Q06455); the localized ciliary-rootlet signal is a specific tissue observation and should not be generalized to diffuse cytoplasm (HPA tissue IHC). |
| Prominent signal in adrenal gland glandular cells or bone-marrow hematopoietic cells. | These cell populations are listed as not detected in tissue IHC (HPA tissue IHC). Check cell identification and background before interpreting signal as RUNX1T1; cross-reactivity or endogenous detection activity can produce misleading chromogen (general IHC practice). |
| Haze or precipitate spans cells and extracellular space, obscuring nuclei and tissue structure. | An unstructured field-wide signal is background rather than the reported low nucleolar or cell-restricted pattern (HPA tissue IHC; general IHC practice). Evaluate the detection-only control and reagent handling before scoring positive cells (general IHC practice). |
| No visible signal in Leydig cells or in ciliary rootlets of an appropriately represented positive tissue. | HPA reports medium staining at those sites, so an absent signal warrants a technical check (HPA tissue IHC). Confirm that the relevant cells and structures are present, then review retrieval, primary-antibody incubation and detection controls (general IHC practice). |
💡Expected RUNX1T1 appearanceCall a section positive when the relevant cells show low nucleolar staining or the reported medium Leydig-cell or ciliary-rootlet signal; widespread unstructured cytoplasmic colour is suspect (HPA tissue IHC; general IHC practice).
| Tissue and cell selection | Brain is tissue enhanced by RNA, yet HPA lists low staining in cerebral-cortex endothelial and cerebellar Purkinje cells, and no staining in hippocampal glia (HPA tissue IHC). Choose controls by the documented IHC cell type rather than assuming every brain cell is strongly positive (HPA tissue IHC). |
| Antibody validation | HPA070951 is Approved for IHC, whereas HPA049852 has no listed IHC status and is Supported for ICC (HPA antibodies). HPA describes low agreement between tissue staining and RNA, so interpret isolated or unexpected IHC signals cautiously (HPA tissue IHC). |
| Protein form and epitope | UniProt lists 6 isoforms, two example phosphoserines, and a full-length 1–604 chain with no signal peptide or propeptide (UniProt Q06455). The supplied evidence gives no antibody epitope or isoform-recognition map; do not infer that a missing or extra cell pattern identifies a particular isoform (UniProt Q06455). |
| IF/ICC Q: Where should fluorescence appear? | A: Predominantly in the nucleoplasm; HPA also lists cytosol as an additional ICC-IF location, and UniProt reports nuclear dots with ATN1 (HPA subcellular ICC-IF; UniProt Q06455). The ICC result does not establish an IHC-P staining rule for diffuse cytoplasm (HPA subcellular ICC-IF; HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected Leydig-cell or ciliary-rootlet signal is absent. | The represented structure may be missing, or retrieval, primary incubation, or detection may have failed (HPA tissue IHC; general IHC practice). | First verify the relevant cells on the section; then check controls and the antibody's validated IHC-P workflow before changing one general IHC variable at a time (general IHC practice). |
| Every cell shows similarly strong colour. | Uniform staining conflicts with the low, cell-specific tissue profile and may reflect nonspecific binding or excessive detection signal (HPA tissue IHC; general IHC practice). | Compare with a primary-omission control; review blocking, washes, primary concentration and chromogen development using the validated IHC-P instructions (general IHC practice). |
| Colour persists in the primary-omission control. | The primary antibody cannot explain signal in that control; endogenous enzyme activity or the detection system is a possible source (general IHC practice). | Inspect the control alongside the sample, then check the detection reagents and use an appropriate endogenous-activity block for the chosen chromogen system (general IHC practice). |
| Signal appears mainly at tissue edges or as granular deposits. | A distribution tied to section geometry or deposits suggests handling or reagent artefact rather than the reported cell-restricted pattern (HPA tissue IHC; general IHC practice). | Inspect section integrity and reagent precipitate, refresh or filter reagents where appropriate, and score only intact areas with identifiable cells (general IHC practice). |
| A proposed negative-control cell population stains. | Adrenal gland glandular cells and bone-marrow hematopoietic cells are not detected in the supplied tissue profile; unexpected signal may be cross-reactivity or background (HPA tissue IHC; general IHC practice). | Verify cell identity, compare primary-omission and positive controls, and avoid treating the unexpected colour as confirmed RUNX1T1 without further validation (general IHC practice). |
| Nuclear staining differs between fields or specimens. | The reported baseline is low in most tissues, and HPA flags low consistency between staining and RNA; weak differences alone may be hard to classify (HPA tissue IHC). | Score the same named cell type and compartment across sections, compare matched controls and record intensity separately from the fraction of positive cells (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing RUNX1T1 staining
A01792-1 · IHCIHC analysis of RUNX1T1/ETO using anti-RUNX1T1/ETO antibody (A01792-1). RUNX1T1/ETO was detected in paraffin-embedded section of human mamma
A01792-1 · IHCIHC analysis of RUNX1T1/ETO using anti-RUNX1T1/ETO antibody (A01792-1). RUNX1T1/ETO was detected in paraffin-embedded section of mouse brain
A01792-1 · IFIF analysis of RUNX1T1 using anti-RUNX1T1 antibody (A01792-1). RUNX1T1 was detected in immunocytochemical section of U20S cell. Enzyme antig
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000079102-RUNX1T1/tissue/cerebral+cortex#imid_19005197
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000079102-RUNX1T1/tissue/colon#imid_19005139
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000079102-RUNX1T1/tissue/liver#imid_19005140
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000079102-RUNX1T1/tissue/kidney#imid_19005125
HPA · ICC-IF HEK293Human Protein Atlas immunofluorescence, HEK293, antibody HPA049852; green = RUNX1T1, blue = nucleus, red = microtubules.
HPA · ICC-IF HEK293Human Protein Atlas immunofluorescence, HEK293, antibody HPA049852; green = RUNX1T1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RUNX1T1 IHC & IF
🧪Run bronchus first and assess staining in ciliated cells at the ciliary rootlets (HPA: Medium in bronchial ciliated cells). Use adrenal gland glandular cells as the negative tissue (HPA: Not detected); on the bronchus slide, assess neighboring cells without ciliary rootlets for background, without assuming they are target-negative.
Positive control tissue: Bronchus (Ciliated cells (ciliary rootlets), HPA Medium)
Negative control tissue: Adrenal gland (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RUNX1T1 in HEK293, U2OS, NIH 3T3, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a concentration-matched rabbit isotype control, and a RUNX1T1 knockout specimen or validated peptide competition control (selected caption: rabbit primary antibody). Block endogenous peroxidase and check endogenous biotin background because the caption uses biotin-based detection with DAB (selected caption: biotinylated secondary, SABC and DAB).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A01792-1 tissue-IHC caption does not state a fixative. The caption reports heat-mediated citrate retrieval at pH 6 for 20 minutes, but does not establish whether retrieval is required; the supplied evidence does not establish whether frozen sections or IF are easier (selected caption: citrate retrieval, 20 minutes). Interpret ciliary-rootlet staining cautiously alongside expected nuclear localization (HPA: ciliary rootlets in bronchus; UniProt Q06455: nucleus).
HPA tissue IHC evidence for RUNX1T1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low 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 |
|---|---|---|---|---|
| Bronchus | Ciliated cells (ciliary rootlets) | Medium | Protein (IHC) | HPA → |
| Fallopian tube | Ciliated cells (ciliary rootlets) | Medium | Protein (IHC) | HPA → |
| Nasopharynx | Ciliated cells (ciliary rootlets) | Medium | Protein (IHC) | HPA → |
| Testis | Leydig cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RUNX1T1 IHC Tips
Troubleshoot RUNX1T1 chromogenic IHC by checking retrieval, nuclear localisation and cell identity before scoring staining intensity.
How should I retrieve RUNX1T1 in paraffin sections when nuclear staining is weak?
Start with heat-mediated retrieval in citrate buffer at pH 6 for 20 minutes (datasheet A01792-1). Use the caption’s 1 μg/mL primary antibody incubation overnight at 4°C as a reference condition when comparing retrieval results (datasheet A01792-1). If staining remains weak, vary heating or cooling conditions on adjacent sections while keeping detection and exposure to DAB constant (standard IHC practice). Score nuclear signal and tissue damage together, since stronger heating can increase staining while reducing morphology or raising background (standard IHC practice). A nucleoplasmic pattern is the expected localisation against which to judge improvement (HPA: supported nucleoplasm).
Can fixation explain absent RUNX1T1 staining in paraffin sections?
Target-specific sensitivity to fixative type or fixation time is unknown: the selected paraffin-section caption does not state the fixative (datasheet A01792-1). Record the fixative and processing history for each specimen, then compare similarly processed sections using the same pH 6 citrate retrieval (standard IHC practice; datasheet A01792-1). Check whether nuclear morphology and staining of an established control antigen are preserved before attributing a negative RUNX1T1 result to fixation (standard IHC practice). Keep primary incubation at the caption’s 1 μg/mL overnight at 4°C during that comparison (datasheet A01792-1). Neither tissue staining patterns nor phosphorylation establish a fixation effect here (HPA: tissue IHC; UniProt Q06455: phosphoserines at residues 41 and 417).
Where should convincing RUNX1T1 staining appear within a positive cell?
Prioritise staining in the nucleoplasm, the supported subcellular location, when evaluating chromogenic sections (HPA: supported nucleoplasm). Discrete nuclear dots can also be biologically plausible because RUNX1T1 colocalises with ATN1 in that pattern (UniProt Q06455: nucleus and ATN1 colocalisation). The additional cytosolic location warrants documenting cytoplasmic signal, but diffuse cytoplasmic staining alone needs independent validation (HPA: additional cytosol; standard IHC practice). Compare compartment-specific signal with a nuclear counterstain at matched magnification and exclude pigment or precipitate outside cell boundaries (standard IHC practice). RUNX1T1 has no transmembrane segment, so a crisp membrane-only pattern conflicts with its recorded topology (UniProt Q06455: topology).
Could isoforms or epitope accessibility change what this antibody detects?
RUNX1T1 has 6 recorded isoforms, so isoform coverage cannot be assumed without an epitope map for the catalog antibody (UniProt Q06455: isoforms). The record identifies a TAFH domain at residues 120–215 and phosphoserines at 41 and 417, but these do not identify this antibody’s epitope (UniProt Q06455: domains and modified residues). If staining differs across specimens, review the antibody’s stated immunogen and test orthogonal expression evidence before assigning the difference to splicing or phosphorylation (standard IHC practice). Compare nuclear localisation and staining in matched processing conditions, because retrieval and detection changes can also alter apparent signal (HPA: supported nucleoplasm; standard IHC practice).
How can I check RUNX1T1 localisation by multiplex immunofluorescence?
Treat IF as a separate assay and first verify that the antibody yields reproducible nucleoplasmic staining against a nuclear counterstain (HPA: supported nucleoplasm; standard IF practice). Multiplex with a validated marker for the cell type under study, using the HPA’s medium-staining Leydig cells or ciliated cells as possible tissue contexts (HPA: tissue IHC). Select fluorophores in channels with low measured tissue autofluorescence, and include single-stain controls to assess bleed-through (standard IF practice). Because the expected epitope is intracellular rather than membrane-exposed, optimise permeabilisation to permit nuclear access while preserving cell structure (UniProt Q06455: nucleus and no transmembrane segment; standard IF practice). Do not transfer the paraffin-section caption’s retrieval or fixation assumptions to IF (datasheet A01792-1: paraffin-section IHC; fixative unreported).
How do I reduce diffuse or patchy background without losing nuclear signal?
Compare a no-primary control with the stained section to identify secondary-reagent, chromogen or tissue-associated background (standard IHC practice). The selected caption used 10% goat serum block, 1 μg/mL primary antibody overnight at 4°C, and biotinylated detection with DAB (datasheet A01792-1). When background is high, titrate primary concentration and incubation conditions on adjacent sections while keeping retrieval at citrate pH 6 (datasheet A01792-1; standard IHC practice). Use a peroxidase block and check a detection-only control; these are general chromogenic workflow checks, not RUNX1T1-specific validation (standard IHC practice). Judge improvement by the nuclear-to-background contrast rather than total brown staining (HPA: supported nucleoplasm; standard IHC practice).
What is a defensible way to quantify RUNX1T1 IHC across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define evaluable cells and a nuclear positivity threshold before scoring, using the supported nucleoplasmic location as the primary compartment (HPA: supported nucleoplasm; standard IHC practice). Report the percentage of positive nuclei and, if intensity is reproducible, an H-score from 0–300 based on percentages at intensity levels 0–3 (standard IHC practice). Normalise counts to the number of evaluable cells in the same cell population, or report positive nuclei per mm² of viable tissue (standard IHC practice). Keep section thickness, citrate pH 6 retrieval and DAB development consistent across groups (datasheet A01792-1; standard IHC practice). Record excluded necrotic, folded and edge regions before comparing scores (standard IHC practice).
How can I distinguish genuine RUNX1T1 positivity from staining artefact?
A convincing result has signal within identifiable nuclei, consistent with supported nucleoplasmic localisation, and exceeds matched negative-control staining (HPA: supported nucleoplasm; standard IHC practice). Nuclear dots may fit reported ATN1 colocalisation, whereas membrane-only staining conflicts with the absence of a transmembrane segment (UniProt Q06455: ATN1 colocalisation and topology). Check cell identity: medium staining is reported in Leydig cells and ciliated-cell rootlets, while HPA flags low agreement between antibody staining and RNA data (HPA: tissue IHC, Approved with low consistency). Reject edge accentuation, necrotic deposits and endogenous peroxidase signal using morphology and no-primary or detection controls (standard IHC practice). Do not interpret a positive stain alone as evidence of an AML1-MTG8/ETO fusion (UniProt Q06455: fusion protein; standard IHC practice).
Boster reagents
Best RUNX1T1 / Protein CBFA2T1 IHC Antibodies
A01792-1 has IHC images from human mammary cancer and mouse brain paraffin sections, plus an IF/ICC image from U20S cells (catalog image captions). Listed reactivity includes human, mouse, and rat (catalog: reactivity).
Real IHC data
Anti-RUNX1T1/ETO Antibody ®
Cat # A01792-1
A01792-1 will render with its human mammary cancer paraffin-section IHC figure (catalog: A01792-1 IHC image caption). The same SKU also has a mouse brain paraffin-section IHC image and a U20S-cell IF/ICC image (catalog: A01792-1 image captions).
Which to pick: For tissue IHC, choose A01792-1: its own captions show paraffin-section staining in human mammary cancer and mouse brain, but do not report the fixative (catalog: A01792-1 IHC image captions). For IF/ICC, A01792-1 is listed for both applications and has a U20S-cell IF image (catalog: A01792-1 applications and IF image caption). For work across species, A01792-1 lists human, mouse, and rat reactivity, though the supplied IHC images cover only human and mouse; no clone designation is supplied.
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q06455 (MTG8_HUMAN, Protein CBFA2T1).
- Human Protein Atlas. RUNX1T1 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. RUNX1T1 subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. RUNX1T1 antibody validation summary (2 antibodies).
- RUNX1T1: a novel predictor of liver metastasis in primary pancreatic endocrine neoplasms. Pancreas 2011 — PMC4732279.
- C/EBPβ regulates homeostatic and oncogenic gastric cell proliferation. Journal of molecular medicine (Berlin, Germany) 2016 — PMC5143359.
- The transcriptional co-repressor Runx1t1 is essential for MYCN-driven neuroblastoma tumorigenesis. Nature communications 2024 — PMC11239676.
- High-resolution melting curve analysis, a rapid and affordable method for mutation analysis in childhood acute myeloid leukemia. Frontiers in pediatrics 2014 — PMC4158872.
- PubMed PMID:8334990 — UniProt-cited evidence.
- PubMed PMID:9661669 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RUNX1T1 (Protein CBFA2T1). Expected localisation Low nucleolar tissue signal (HPA tissue IHC); nuclear dots (UniProt). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A01792-1). Validated IHC antibody: A01792-1 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Bronchus (Ciliated cells (ciliary rootlets)); negative Adrenal gland. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human mammary cancer tissue; fixative not specified (datasheet A01792-1); 2. Fixation: Image fixative and duration unreported (datasheet A01792-1); 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: Citrate pH 6 , 20 min (datasheet A01792-1); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A01792-1); 8. Primary antibody: Rabbit anti-RUNX1T1, 0.5-1μg/ml (datasheet A01792-1); 9. Primary incubation: Overnight at 4 °C (datasheet A01792-1). Watch for: Fixation: Keep fixation conditions consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Tissue staining has low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: 6 isoforms; check epitope coverage across variants (UniProt); Regulation: Brain-enhanced RNA expression (HPA RNA). Evidence: UniProt Q06455, HPA tissue IHC · Approved, HPA ICC-IF · nucleoplasm., Datasheet A01792-1, PMC5143359, PMC11239676. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RUNX1T1/immunohistochemistry-ihc-guide.