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SART3 / Spliceosome associated factor 3, U4/U6 recycling protein · IHC design guide
Design Immunohistochemistry for SART3
Plan chromogenic SART3 IHC in paraffin sections using mainly nuclear staining as the expected tissue pattern (HPA tissue IHC). Breast glandular cells and bone marrow hematopoietic cells provide high-staining references, while low consistency between staining and RNA expression warrants caution when scoring (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
SART3 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 | Mainly nuclear tissue staining (HPA tissue IHC) | |
| Staining pattern | Ubiquitous, mainly nuclear staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A03973-2) | |
| Positive control | Adrenal gland+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 conditions consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA expression show low consistency (HPA tissue IHC) | |
| Regulation | Ubiquitously expressed (UniProt) | |
| Isoform / epitope | 4 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended SART3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet A03973-2) is accompanied by published SART3 staining conditions for human HCC tissue (PMC5256867; PMC10496991).
| Sample | Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A03973-2) |
| Fixation | Image fixative and duration unreported (datasheet A03973-2); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A03973-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A03973-2) |
| Primary antibody | Rabbit anti-SART3, 2-5 μg/ml (datasheet A03973-2) |
| Primary incubation | Overnight at 4 °C (datasheet A03973-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A03973-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | SART3-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Ubiquitous mainly nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval (datasheet A03973-2). The pressure-cooker method specifies 1–4 min but no retrieval buffer (PMC5256867).
Section 2
What Is the Expected SART3 Staining Pattern?
SART3 is expected mainly in nuclei, consistent with UniProt nucleoplasm, Cajal body and nuclear speckle annotations; UniProt also lists cytoplasm and no transmembrane segment (UniProt Q15020). HPA describes ubiquitous, mainly nuclear tissue staining, with high staining in several glandular, hematopoietic, epithelial and neuronal cell populations (HPA tissue IHC). Its tissue IHC reliability is Approved, with low consistency between staining and RNA expression (HPA tissue IHC).
| Predominantly nuclear staining in an HPA high-staining population, such as bone marrow hematopoietic cells or breast glandular cells (HPA tissue IHC). | This fits the reported mainly nuclear tissue pattern (HPA tissue IHC) and UniProt nuclear locations (UniProt Q15020). Compare cell identity and nuclear localisation together; a positive-looking region alone does not establish antibody specificity (standard IHC interpretation). |
| Predominantly cytoplasmic staining with weak or absent nuclear staining. | This departs from HPA's mainly nuclear tissue pattern (HPA tissue IHC). UniProt also lists cytoplasm, so some cytoplasmic signal cannot be dismissed categorically (UniProt Q15020). Check morphology and controls before treating a cytoplasm-dominant result as SART3-specific (standard IHC interpretation). |
| Strong staining in cells other than the annotated high-staining population within a sampled tissue (HPA tissue IHC). | Consider cross-reactivity or endogenous detection activity, especially if the signal also appears in a reagent control (standard IHC practice). SART3 is ubiquitously expressed, so an unlisted cell type is not, by itself, a valid negative control (UniProt Q15020; HPA tissue IHC). |
| Diffuse colour over nuclei, cytoplasm and extracellular areas, without clear cellular boundaries. | This is harder to reconcile with HPA's mainly nuclear pattern (HPA tissue IHC). Non-specific reagent binding, excess detection signal or endogenous enzyme activity can create broad chromogenic background; inspect controls before scoring individual nuclei (standard IHC practice). |
| No convincing nuclear signal in an HPA high-staining population, such as bronchial respiratory epithelial cells (HPA tissue IHC). | First assess section quality, cell identification, controls and assay conditions (standard IHC practice). HPA reports high staining in that population, but its Approved tissue profile has low consistency with RNA expression, so a single negative section needs cautious interpretation (HPA tissue IHC). |
💡Expected SART3 appearanceCall positive when identifiable cells show predominantly nuclear chromogenic staining, especially in an HPA high-staining population (HPA tissue IHC); strong diffuse or nucleus-sparing colour is a warning pattern to investigate with controls (standard IHC interpretation).
| Subcellular reference | HPA reports ubiquitous mainly nuclear tissue staining, while UniProt lists nucleoplasm, Cajal bodies, nuclear speckles and cytoplasm (HPA tissue IHC; UniProt Q15020). Nuclear predominance is the practical IHC expectation; cytoplasmic signal needs context. |
| Tissue benchmarks | HPA calls adrenal and breast glandular cells, bone marrow hematopoietic cells, and several neuronal populations High; lung alveolar cells, oral squamous epithelial cells and liver cholangiocytes are Low (HPA tissue IHC). Low means weaker reported staining, not a validated absence. |
| Antibody evidence | The HPA tissue profile is Approved but has low agreement between antibody staining and RNA expression (HPA tissue IHC). Among listed antibodies, HPA044322 is IHC Approved; HPA028840 has ICC Supported status with no listed IHC status (HPA antibodies). |
| Molecular context | SART3 has four reported isoforms and no transmembrane segment or signal peptide (UniProt Q15020). The supplied record gives no antibody epitope, so isoform recognition cannot be inferred; topology supports evaluating intracellular compartments rather than a membrane rim (UniProt Q15020). |
| Preparation and detection | The supplied UniProt and HPA records give no SART3-specific fixation sensitivity or antigen retrieval condition. If adjusting retrieval or detection, treat those changes as general IHC optimisation and judge them against morphology and controls (standard IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| Known high-staining cells show no nuclear signal (HPA tissue IHC). | The cells may be misidentified, the section may be compromised, or the staining workflow may have failed (standard IHC practice). | Confirm tissue morphology and run a suitable assay control; review retrieval, antibody dilution and detection conditions as general IHC checks (standard IHC practice). Do not infer SART3-specific fixation sensitivity from HPA staining levels. |
| Colour is mainly cytoplasmic or outlines membranes. | A membrane rim conflicts with the reported mainly nuclear IHC pattern and UniProt's lack of a transmembrane segment (HPA tissue IHC; UniProt Q15020). UniProt does list cytoplasm (UniProt Q15020). | Recheck nuclear counterstain, cellular boundaries and reagent controls; score any cytoplasmic signal separately from nuclear signal (standard IHC practice). |
| Unexpected cells stain more strongly than the annotated population (HPA tissue IHC). | Cross-reactivity or endogenous enzyme activity is possible, but ubiquitous expression limits claims that a cell type must be negative (UniProt Q15020; standard IHC practice). | Compare the annotated cells in the same section and inspect reagent controls; use an independent specificity check before assigning the unexpected signal to SART3 (standard IHC practice). |
| Broad background obscures nuclei. | Non-specific binding, excessive detection signal or endogenous peroxidase can produce diffuse chromogenic colour (standard IHC practice). | Review blocking, washing, antibody concentration and endogenous-enzyme controls; retain conditions that preserve interpretable nuclear staining and morphology (standard IHC practice). |
| A low-staining population appears negative. | HPA labels lung alveolar cells, oral squamous epithelial cells and liver cholangiocytes Low, rather than absent (HPA tissue IHC). | Interpret alongside a high-staining population and assay controls; avoid calling a Low population a confirmed negative or using it alone to reject the assay (HPA tissue IHC; standard IHC practice). |
| What should IF/ICC show? | HPA supports nucleoplasmic localisation in ICC-IF and lists HAP1 and HEL image sets; UniProt additionally lists other nuclear sites and cytoplasm (HPA subcellular; UniProt Q15020). | Use the separate IF/ICC guide for that application. Compare any IF image with the supported nucleoplasmic reference; do not transfer an IF pattern or protocol setting directly into IHC-P scoring (HPA subcellular; standard assay interpretation). |
Experiment evidence
IHC & IF images showing SART3 staining
A03973-2 · IHCIHC analysis of SART3 using anti-SART3 antibody (A03973-2). SART3 was detected in a paraffin-embedded section of human breast cancer tissue.
A03973-2 · IHCIHC analysis of SART3 using anti-SART3 antibody (A03973-2). SART3 was detected in a paraffin-embedded section of human colorectal adenocarci
A03973-2 · IFIF analysis of SART3 using anti-SART3 antibody (A03973-2) and anti-Tubulin Alpha antibody (M03989-3). SART3 was detected in immunocytochemic
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000075856-SART3/tissue/cerebral+cortex#imid_12734907
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000075856-SART3/tissue/colon#imid_12734833
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000075856-SART3/tissue/liver#imid_12734826
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000075856-SART3/tissue/kidney#imid_12734827
HPA · ICC-IF HAP1Human Protein Atlas immunofluorescence, HAP1, antibody HPA028840; green = SART3, blue = nucleus, red = microtubules.
HPA · ICC-IF HAP1Human Protein Atlas immunofluorescence, HAP1, antibody HPA028840; green = SART3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for SART3 IHC & IF
🧪Run breast first: its glandular cells should stain for SART3 (HPA: breast glandular cells, High). HPA detects SART3 in all 45 scored tissues, so no negative tissue is established; use no-primary and isotype controls, and treat cells without nuclear staining on the positive slide only as background references, not proven SART3-negative cells (HPA: no negative rows; UniProt Q15020: ubiquitously expressed).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: SART3 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 SART3 in HAP1, HEL, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide, a concentration-matched rabbit IgG isotype control, and a SART3 knockout specimen as a biological negative (caption: rabbit primary antibody; standard IHC practice). Quench endogenous peroxidase and check blood-rich areas for residual chromogenic signal (caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected paraffin-section caption does not state the fixative (caption: fixative not stated). Paraffin IHC is documented with heat-mediated EDTA retrieval at pH 8.0; frozen sections are undocumented here, while HAP1 and HEL ICC-IF images support IF/ICC feasibility without establishing that it is easier (caption: EDTA retrieval; HPA subcellular: HAP1 and HEL). In breast sections, assess expected nuclear staining against residual peroxidase signal and diffuse DAB background (UniProt Q15020: nucleoplasm; standard IHC practice).
HPA tissue IHC evidence for SART3
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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: SART3 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced SART3 IHC Tips
Troubleshoot SART3 staining by checking retrieval, nuclear localisation, and background before comparing signal across paraffin sections.
What retrieval should I try first when SART3 staining is weak?
Start with heat-mediated retrieval in EDTA at pH 8.0 (datasheet A03973-2). The catalog antibody stained a paraffin-embedded human breast cancer section after that retrieval, followed by 2 μg/ml primary antibody overnight at 4°C (caption A03973-2). If staining remains weak, compare a longer or shorter heating interval on adjacent sections while keeping the primary concentration and detection conditions fixed (standard IHC practice). Include a section processed without primary antibody to reveal retrieval-related background (standard IHC practice). Judge any gain by clearer nuclear signal rather than increased diffuse staining, because SART3 is predominantly nuclear in tissue IHC (HPA tissue IHC).
Could fixation explain variable nuclear SART3 staining between blocks?
The selected tissue caption identifies a paraffin-embedded section but does not state its fixative or fixation duration (caption A03973-2). Target-specific SART3 sensitivity to fixation is therefore unknown; neither its intracellular localisation nor its reported modifications establish a fixation effect (UniProt Q15020). Record each block’s fixative, duration, and processing history before comparing stain intensity (standard IHC practice). When those details differ, stain adjacent sections in the same run using EDTA retrieval at pH 8.0 and the same detection conditions (datasheet A03973-2; standard IHC practice). Interpret remaining intensity differences cautiously until comparable processing and an appropriate positive control support them (standard IHC practice).
How should I assess cytoplasmic or punctate SART3 staining?
Score nuclear staining separately from cytoplasmic staining: tissue IHC describes SART3 as ubiquitous and mainly nuclear (HPA tissue IHC). Nucleoplasmic localisation is supported by cellular imaging, while UniProt also lists Cajal bodies, nuclear speckles, and cytoplasm (HPA subcellular; UniProt Q15020 localisation). Check whether puncta lie within counterstained nuclei before calling them nuclear structures (standard IHC practice). Diffuse cytoplasmic DAB alone deserves review of background, counterstain, and the no-primary control rather than automatic assignment as SART3 (standard IHC practice). Compare localisation in a high-staining reference cell population, such as breast glandular cells, processed alongside the study sections (HPA tissue IHC; standard IHC practice).
Could isoforms or epitope accessibility change the IHC pattern?
SART3 has 4 annotated isoforms and two RNA recognition motifs at residues 704–782 and 801–878 (UniProt Q15020). The supplied antibody caption does not identify its epitope, so staining cannot be assigned to a particular isoform or modified residue (caption A03973-2; UniProt Q15020). Compare staining after the documented EDTA retrieval at pH 8.0 under otherwise matched conditions before attributing weak signal to epitope masking (datasheet A03973-2; standard IHC practice). If isoform discrimination matters, obtain the immunogen location and evaluate its overlap with each sequence using an independently validated approach (standard IHC practice). Keep isoform claims out of the IHC score until that specificity is established (standard IHC practice).
How can IF help resolve uncertain SART3 IHC localisation?
Use IF as a separate localisation check, with a nuclear counterstain and a marker identifying the expected cell type in the specimen (standard IF practice). Choose spectrally separated fluorophores and consider a far-red SART3 channel when tissue autofluorescence obscures shorter wavelengths (standard IF practice). SART3 is intracellular and has no transmembrane segment, so permeabilisation must allow antibody access to its nuclear epitope; optimise it against preserved morphology (UniProt Q15020 topology and localisation; standard IF practice). Compare nuclear signal with the supported nucleoplasmic pattern, while allowing for other reported compartments (HPA subcellular; UniProt Q15020 localisation). The supplied caption documents chromogenic tissue IHC, not an IF preparation or IF fixation condition (caption A03973-2).
What should I check when DAB obscures SART3 nuclei?
Start with a no-primary section and inspect whether brown signal follows nuclei, section edges, damaged areas, or endogenous pigment (standard IHC practice). Block endogenous peroxidase before HRP detection, and verify that blocking and washes are adequate for the tissue (standard chromogenic IHC practice). The documented tissue staining used 10% goat serum, 2 μg/ml primary antibody overnight at 4°C, an HRP-linked detection system, and DAB (caption A03973-2). If background persists, titrate primary antibody below the documented concentration and compare signal with background on adjacent sections (standard IHC practice). Treat diffuse DAB cautiously because tissue IHC describes SART3 as mainly nuclear (HPA tissue IHC).
How should I quantify nuclear SART3 across tissue samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and scoring region before reading slides, then score nuclear SART3 separately from cytoplasmic signal (standard IHC practice; HPA tissue IHC). Report the percentage of positive nuclei and intensity, or calculate a nuclear H-score on a 0–300 scale (standard IHC practice). For sparse populations, positive nuclei per mm² can be useful if the analysed area and cell selection are recorded (standard IHC practice). Normalise comparisons to eligible cells or tissue area, and keep retrieval, antibody concentration, DAB development, and imaging conditions consistent (standard IHC practice). HPA reports low consistency between antibody staining and RNA expression, so avoid treating IHC intensity as a direct RNA measurement (HPA tissue IHC).
How do I distinguish convincing SART3 staining from artefact?
A convincing result shows interpretable nuclear staining in intact cells, consistent with the mainly nuclear tissue profile and supported nucleoplasmic localisation (HPA tissue IHC; HPA subcellular). High staining in breast glandular cells offers a reference pattern, while staining is reported as low in lung alveolar cells, oral squamous epithelial cells, and liver cholangiocytes (HPA tissue IHC). Check unexpected compartment or cell patterns against morphology and matched controls before assigning biological meaning (standard IHC practice). Exclude edge accentuation, necrotic regions, pigment, and endogenous peroxidase signal from positive scoring (standard IHC practice). Because HPA reports low staining–RNA consistency, confirm consequential biological interpretations with an independent method (HPA tissue IHC; standard IHC practice).
Boster reagents
Best SART3 / Spliceosome associated factor 3, U4/U6 recycling protein IHC Antibodies
The IHC-validated anti-SART3 antibody has image data from human paraffin tissue sections and IF data from A549 cells and human paraffin tissue sections (catalog: A03973-2 image captions).
Real IHC data
Anti-SART3 Antibody ®
Cat # A03973-2
A03973-2 has IHC image data from paraffin sections of human breast cancer, colorectal adenocarcinoma and laryngeal squamous cell carcinoma (catalog: A03973-2 IHC image captions). It also has IF image data from A549 cells and a paraffin section of human esophageal squamous carcinoma (catalog: A03973-2 IF image captions).
Which to pick: For tissue IHC, choose A03973-2: its paraffin-section image captions document EDTA pH 8.0 retrieval, 2 μg/ml primary antibody and DAB detection; the fixative is unreported (catalog: A03973-2 IHC image captions). For IF/ICC, A03973-2 lists both applications and has corresponding image data (catalog: A03973-2 applications and IF image captions). No cross-species option is documented here; A03973-2 lists human reactivity only, and its clonality is unspecified (catalog: A03973-2 reactivity and clone fields).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q15020 (SART3_HUMAN, Spliceosome associated factor 3, U4/U6 recycling protein).
- Human Protein Atlas. SART3 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. SART3 subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. SART3 antibody validation summary (2 antibodies).
- Cellular Immune Responses for Squamous Cell Carcinoma Antigen Recognized by T Cells 3 in Patients with Hepatocellular Carcinoma. PloS one 2017 — PMC5256867.
- SART3, regulated by p53, is a biomarker for diagnosis, prognosis and immune infiltration in hepatocellular carcinoma. Aging 2023 — PMC10496991.
- PubMed PMID:10463607 — UniProt-cited evidence.
- PubMed PMID:11959860 — UniProt-cited evidence.
- PubMed PMID:8590280 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for SART3 (Spliceosome associated factor 3, U4/U6 recycling protein). Expected localisation Mainly nuclear tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A03973-2). Validated IHC antibody: A03973-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A03973-2); 2. Fixation: Image fixative and duration unreported (datasheet A03973-2); 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: EDTA pH 8.0 (datasheet A03973-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A03973-2); 8. Primary antibody: Rabbit anti-SART3, 2-5 μg/ml (datasheet A03973-2); 9. Primary incubation: Overnight at 4 °C (datasheet A03973-2). Watch for: Fixation: Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining and RNA expression show low consistency (HPA tissue IHC); Isoform / epitope: 4 isoforms; epitope coverage is unspecified (UniProt); Regulation: Ubiquitously expressed (UniProt). Evidence: UniProt Q15020, HPA tissue IHC · Approved, HPA ICC-IF · nucleoplasm., Datasheet A03973-2, PMC5256867, PMC10496991. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/SART3/immunohistochemistry-ihc-guide.