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SF3B3 / Splicing factor 3B subunit 3 · IHC design guide
Design Immunohistochemistry for SF3B3
Plan SF3B3 chromogenic IHC around its general nuclear tissue pattern (HPA tissue IHC). The catalog antibody A08240-3 has a reported IHC concentration of 2–5 μg/mL (datasheet).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
SF3B3 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 | General nuclear staining in tissue sections (HPA tissue IHC) | |
| Staining pattern | Nuclear staining across many cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A08240-3) | |
| Positive control | Adipose tissue+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Soft-tissue fibroblasts show low staining (HPA tissue IHC) | |
| Regulation | No specific regulator reported (UniProt) | |
| Isoform / epitope | 3 isoforms; epitope differences unreported (UniProt) |
Section 1
Recommended SF3B3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet A08240-3) is accompanied by one published paraffin-section protocol (PMC7999425).
| Sample | Paraffin-embedded human spleen tissue; fixative not specified (datasheet A08240-3) |
| Fixation | Image fixative and duration unreported (datasheet A08240-3); 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 A08240-3); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A08240-3) |
| Primary antibody | Rabbit anti-SF3B3, 2-5 μg/ml (datasheet A08240-3) |
| Primary incubation | Overnight at 4 °C (datasheet A08240-3) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A08240-3) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | SF3B3-positive staining in adipocytes of adipose tissue (HPA tissue IHC: High). HPA tissue profile: General nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval (datasheet A08240-3); compare the published Tris/EDTA pH 9.0 condition during optimization (PMC7999425).
Section 2
What Is the Expected SF3B3 Staining Pattern?
SF3B3 is a nuclear spliceosome protein with no transmembrane segment (UniProt Q15393: location, function, topology). In paraffin-section IHC, expect a general nuclear pattern across many cell types (HPA tissue IHC: Supported; general nuclear expression). HPA reports high staining in adipocytes, glandular cells, hematopoietic cells, respiratory epithelial cells, glial cells and Purkinje cells, while soft-tissue fibroblasts are low (HPA tissue IHC).
| Nuclear chromogen is clear in adipocytes, glandular cells or hematopoietic cells. | This matches the reported compartment and high-staining cell types (UniProt Q15393: nucleus; HPA tissue IHC: general nuclear expression; High in these cell types). Judge intensity against other cells in the same section; HPA's levels describe observed staining, not a universal cutoff. |
| Strong cytoplasmic staining dominates while nuclei remain pale. | This conflicts with the reported nuclear pattern (UniProt Q15393: nucleus; HPA tissue IHC: general nuclear expression). Consider nonspecific binding or chromogen background before calling it SF3B3. A weak cytoplasmic tint alone does not establish a new localisation. |
| An unexpected cell population stains strongly, with little nuclear contrast. | Check its identity and compartment before interpreting the signal. Cross-reactivity or endogenous detection activity can produce misleading staining (general IHC practice). HPA reports low staining in soft-tissue fibroblasts, but low is not equivalent to absent (HPA tissue IHC: Low in fibroblasts). |
| Chromogen spreads across tissue or blank areas without defined nuclei. | Treat this as background until staining can be assigned to cells. Inadequate blocking, residual detection activity or excess reagent can cause diffuse signal (general IHC practice). The expected result has nuclear contrast (HPA tissue IHC: general nuclear expression). |
| Nuclei are unstained in a section expected to contain high-staining cells. | Adipocytes, bone-marrow hematopoietic cells and bronchial respiratory epithelial cells are reported High (HPA tissue IHC). If those cells are present and identifiable, first check the IHC run and controls (general IHC practice); one negative slide does not overturn the reported pattern. |
💡Expected SF3B3 appearanceCall a result positive when identifiable cells show nuclear chromogen, with clear signal in HPA High populations; cytoplasm-only colour or diffuse deposit without nuclear definition is suspect (UniProt Q15393: nucleus; HPA tissue IHC: general nuclear expression and reported levels; general IHC practice).
| Cell type and tissue | HPA describes low tissue specificity at the RNA level and general nuclear protein staining (HPA tissue IHC). Use its reported High cell types as practical positive references, including adrenal glandular cells and cerebellar Purkinje cells; do not treat the Low fibroblast entry as a negative control (HPA tissue IHC). |
| Compartment and protein topology | SF3B3 belongs to the nuclear U2 snRNP spliceosome and has no transmembrane segment or signal peptide (UniProt Q15393: function, subcellular location, topology, processing). These annotations support nuclear interpretation; they do not establish how fixation or antigen retrieval affects staining. |
| Isoforms and antibody recognition | UniProt lists 3 SF3B3 isoforms (UniProt Q15393: isoforms). The supplied record gives no antibody epitope or isoform-specific IHC result, so apparent differences between tissues cannot be assigned to an isoform from staining alone. |
| Antibody validation | HPA lists IHC as Supported for HPA042986 and ICC as Supported for HPA041134 (HPA antibodies). These are application-specific entries; the ICC rating does not validate an IHC result. The supplied HPA tissue profile rates its IHC evidence Supported for consistency with RNA expression (HPA tissue IHC). |
| Q: What should IF/ICC show? | A: HPA supports nucleoplasm, nucleoli and nucleoli-rim localisation, with images listed for MCF-7, SiHa and U2OS (HPA subcellular ICC-IF). These finer IF compartments need not be separately resolved in chromogenic paraffin IHC (general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| No nuclear signal in a reported High cell population | The run may have failed, or the chosen IHC conditions may be unsuitable (general IHC practice). | Verify cell identity, positive and negative controls, retrieval, antibody dilution and detection steps (general IHC practice). The HPA High designation is an observed reference, not a guaranteed result under every protocol (HPA tissue IHC). |
| Predominantly cytoplasmic staining | Nonspecific binding or background is possible (general IHC practice); the reported location is nuclear (UniProt Q15393; HPA tissue IHC). | Compare with a negative control and inspect nuclear detail; adjust blocking or antibody concentration if background persists (general IHC practice). |
| Strong signal in cells expected to stain weakly | Cell identification, cross-reactivity or endogenous detection activity may explain the discrepancy (general IHC practice). | Confirm the cell type and nuclear compartment; check detection controls before comparing with HPA's Low soft-tissue fibroblast entry (HPA tissue IHC; general IHC practice). |
| Diffuse colour obscures nuclei | Nonspecific reagent binding, inadequate blocking or excess chromogen may reduce contrast (general IHC practice). | Review negative-control staining, blocking, reagent concentration and chromogen development; score only identifiable nuclear signal (general IHC practice; HPA tissue IHC: general nuclear expression). |
| Nuclear signal varies between tissue regions | Different cell populations can have different reported staining levels; a technical gradient is also possible (HPA tissue IHC; general IHC practice). | Compare like cell types across regions and inspect controls and section quality before assigning a biological difference (general IHC practice). |
Experiment evidence
IHC & IF images showing SF3B3 staining
A08240-3 · IHCIHC analysis of SF3B3 using anti-SF3B3 antibody (A08240-3). SF3B3 was detected in a paraffin-embedded section of human spleen tissue. Heat m
A08240-3 · IHCIHC analysis of SF3B3 using anti-SF3B3 antibody (A08240-3). SF3B3 was detected in a paraffin-embedded section of human breast cancer tissue.
A08240-3 · IFIF analysis of SF3B3 and Tubulin alpha using anti-SF3B3 antibody (A08240-3) and anti-Tubulin alpha antibody (M03989-3). SF3B3 and Tubulin al
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000189091-SF3B3/tissue/cerebral+cortex#imid_12365649
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000189091-SF3B3/tissue/colon#imid_12204807
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000189091-SF3B3/tissue/liver#imid_12204824
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000189091-SF3B3/tissue/kidney#imid_12204809
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA041134; green = SF3B3, blue = nucleus, red = microtubules.
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA041134; green = SF3B3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for SF3B3 IHC & IF
🧪Run bone marrow first; hematopoietic cell nuclei should stain (HPA: High in bone marrow hematopoietic cells; UniProt Q15393: nucleus). HPA detects SF3B3 in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and expect anucleate erythrocytes on the positive slide to lack nuclear staining (HPA: no negative tissue rows; standard IHC practice).
Positive control tissue: Adipose tissue (Adipocytes, HPA High)
Negative control tissue: None in HPA: SF3B3 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 SF3B3 in MCF-7, SiHa, U2OS, with annotated localisation: Nucleoplasm (supported), Nucleoli (supported), Nucleoli rim (supported) (HPA subcellular).
Technical controls: Include a secondary-only slide with no primary antibody, a rabbit IgG isotype control matched to the primary antibody’s clonality, and an identically processed SF3B3 knockout sample or peptide-blocked primary if the immunizing peptide is available (selected-SKU caption: rabbit primary; standard IHC practice). Quench endogenous peroxidase in bone marrow and check residual staining before interpreting DAB signal (HPA: bone marrow hematopoietic cells; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU tissue-IHC caption). That caption uses heat retrieval in EDTA at pH 8.0, but does not establish whether retrieval is required; frozen-section suitability and whether IF is easier are unreported (selected-SKU tissue-IHC caption). In bone marrow, endogenous peroxidase can complicate chromogenic scoring, so interpret nuclear staining against the quenched controls (HPA: bone marrow hematopoietic cells; standard IHC practice).
HPA tissue IHC evidence for SF3B3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — 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 |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Adipocytes | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: SF3B3 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced SF3B3 IHC Tips
Use nuclear staining, the selected antibody’s spleen section, and matched controls to troubleshoot SF3B3 chromogenic IHC (UniProt Q15393; datasheet A08240-3).
Which antigen retrieval should I start with for SF3B3 paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for SF3B3 paraffin sections (datasheet A08240-3). The selected antibody produced staining in a human spleen paraffin section after this retrieval, followed by 2 μg/ml primary antibody overnight at 4°C (datasheet A08240-3). If nuclear staining is weak, vary heating duration within the instrument’s recommended range while keeping section thickness, antibody concentration, and detection conditions matched (standard IHC practice). Compare tissue morphology and nuclear signal across those sections, because excessive heating can damage morphology and complicate interpretation (standard IHC practice; UniProt Q15393 subcellular location).
How should I troubleshoot fixation when SF3B3 nuclear staining is weak?
The selected spleen image documents a paraffin section but does not state its fixative, so SF3B3-specific sensitivity to fixation is unknown (datasheet A08240-3). Record fixative, fixation duration, processing history, and section age for each specimen before comparing staining intensity (standard IHC practice). Process a known positive tissue alongside the test sections, then apply the documented EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody as a consistent starting point (datasheet A08240-3; standard IHC practice). If staining differs between batches, assess preservation and retrieval together; neither the nuclear location nor the two annotated phosphorylation sites establishes a fixation effect (UniProt Q15393; standard IHC practice).
Where should convincing SF3B3 staining appear in tissue sections?
Expect predominantly nuclear staining: SF3B3 is annotated in the nucleus, and tissue IHC shows general nuclear expression (UniProt Q15393 subcellular location; HPA tissue IHC). Nucleoplasm, nucleoli, and the nucleolar rim are supported locations in cell imaging, although those structures may be difficult to resolve with chromogenic tissue IHC (HPA subcellular; standard IHC practice). Compare DAB signal with a nuclear counterstain and inspect several intact cells before calling a compartment positive (standard IHC practice). Diffuse extracellular or purely cytoplasmic color deserves a control check, especially if it persists without primary antibody, because it does not match the reported localisation (UniProt Q15393 subcellular location; standard IHC practice).
Could isoforms or epitope accessibility explain inconsistent SF3B3 staining?
SF3B3 has 3 annotated isoforms, but the supplied antibody caption does not identify its epitope or establish which isoforms it detects (UniProt Q15393 isoforms; datasheet A08240-3). The protein has no annotated transmembrane segment, and phosphorylation is annotated at residues 156 and 1200; neither observation establishes an effect on this antibody’s binding (UniProt Q15393 topology and modified residues). Keep retrieval at EDTA pH 8.0 when first comparing specimens, and check that the same nuclear compartment is scored throughout (datasheet A08240-3; HPA tissue IHC; standard IHC practice). Treat an isoform-specific explanation as unresolved until the antibody epitope and relevant isoform sequences can be compared (UniProt Q15393 isoforms; standard IHC practice).
How can IF help check an ambiguous SF3B3 IHC pattern?
Use IF as a separate localisation check, with SF3B3 assessed against a nuclear stain and a marker for the expected cell population; HPA reports high staining in bone marrow hematopoietic cells (HPA tissue IHC; standard IF practice). Choose a spectrally separated fluorophore, considering a far-red channel when the specimen shows strong shorter-wavelength autofluorescence, and include single-color controls for multiplex imaging (standard IF practice). SF3B3 is nuclear and has no transmembrane segment, so permeabilisation must allow antibody access to the nuclear epitope (UniProt Q15393 topology and subcellular location; standard IF practice). HPA cell images support nucleoplasmic, nucleolar, and nucleolar-rim localisation; compare these patterns with the nuclear chromogenic signal without assuming identical image resolution (HPA subcellular; standard IHC/IF practice).
What should I check when DAB obscures SF3B3 nuclear staining?
First compare a no-primary control with the stained section to identify color arising from detection reagents or tissue rather than primary-antibody binding (standard IHC practice). Include an endogenous peroxidase block before HRP/DAB detection, and check whether pigment or residual enzyme activity remains in the control (standard IHC practice). The selected spleen caption used 10% goat serum blocking, a peroxidase-conjugated secondary, and DAB, providing a documented starting workflow for this antibody (datasheet A08240-3). If background remains high, review blocking, wash stringency, primary concentration, and development time one variable at a time while preserving interpretable nuclear morphology (standard IHC practice; HPA tissue IHC).
How should I quantify SF3B3 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define positive staining by nuclear DAB signal and apply the same threshold to every slide, with the nuclear counterstain used to identify evaluable cells (HPA tissue IHC; standard IHC practice). Report the percentage of positive nuclei and, if intensity matters, an H-score from 0–300 calculated as the sum of each intensity category multiplied by its percentage of cells (standard IHC practice). Normalise cell counts to the number of evaluable nuclei within the same annotated tissue compartment, and exclude folds, necrosis, and poorly preserved edges consistently (standard IHC practice). Record exposure to retrieval and DAB development conditions because technical variation can alter apparent nuclear intensity across sections (standard IHC practice).
How do I distinguish true SF3B3 staining from tissue artefact?
A credible result places DAB in intact nuclei and is consistent across comparable cells; SF3B3 is nuclear, and HPA describes general nuclear tissue expression (UniProt Q15393 subcellular location; HPA tissue IHC; standard IHC practice). Check the relevant cell compartment against the counterstain: HPA reports high staining in several populations, including bone marrow hematopoietic cells, so cell identity matters when comparing tissues (HPA tissue IHC; standard IHC practice). Treat staining confined to section edges, necrotic areas, or acellular deposits as suspect, and compare it with a no-primary control (standard IHC practice). Residual endogenous peroxidase can also produce DAB color; interpret intensity only after control staining and morphology support a nuclear signal (standard IHC practice).
Boster reagents
Best SF3B3 / Splicing factor 3B subunit 3 IHC Antibodies
A08240-3 has IHC images from human paraffin sections and IF images from PC-3 cells, human ovarian cancer, and rat brain (catalog image captions). M08240-1 lists IHC and IF/ICC (catalog applications).
Real IHC data
Anti-SF3B3 Antibody ®
Cat # A08240-3
A08240-3 shows IHC in paraffin-embedded human spleen, breast cancer, colon adenocarcinoma, and larynx squamous cell carcinoma, plus IF in PC-3 cells, human ovarian cancer, and rat brain (catalog image captions). M08240-1 lists IHC and ICC/IF and human, mouse, and rat reactivity, but has no supplied IHC or IF image captions (catalog applications/reactivity/image alts).
Which to pick: For tissue IHC, choose A08240-3: its own human spleen caption documents a paraffin section, EDTA retrieval at pH 8.0, and 2 μg/ml primary antibody overnight at 4°C; the fixative is unreported (A08240-3 IHC image caption). For IF/ICC, A08240-3 has images from PC-3 cells and human ovarian cancer and rat brain sections (A08240-3 IF image captions); M08240-1 is a rabbit monoclonal, clone 19S30, that lists ICC/IF but has no supplied image captions (catalog clone/applications/image alts). For cross-species work, both list human, mouse, and rat reactivity (catalog reactivity), while A08240-3 also has a rat brain IF image (A08240-3 IF image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q15393 (SF3B3_HUMAN, Splicing factor 3B subunit 3).
- Human Protein Atlas. SF3B3 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. SF3B3 subcellular location (ICC-IF): Localized to the nucleoplasm, nucleoli and nucleoli rim..
- Human Protein Atlas. SF3B3 antibody validation summary (2 antibodies).
- Alternative Splicing of EZH2 pre-mRNA by SF3B3 Contributes to the Tumorigenic Potential of Renal Cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 2017 — PMC5440213.
- SF3B3-regulated mTOR alternative splicing promotes colorectal cancer progression and metastasis. Journal of experimental & clinical cancer research : CR 2024 — PMC11047005.
- Ambiguity about Splicing Factor 3b Subunit 3 (SF3B3) and Sin3A Associated Protein 130 (SAP130). Cells 2021 — PMC7999425.
- Depleted HDAC3 attenuates hyperuricemia-induced renal interstitial fibrosis via miR-19b-3p/SF3B3 axis. Cell cycle (Georgetown, Tex.) 2022 — PMC8942505.
- PubMed PMID:10490618 — UniProt-cited evidence.
- PubMed PMID:7584026 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for SF3B3 (Splicing factor 3B subunit 3). Expected localisation General nuclear staining in tissue sections (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A08240-3). Validated IHC antibody: A08240-3 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adipose tissue (Adipocytes); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human spleen tissue; fixative not specified (datasheet A08240-3); 2. Fixation: Image fixative and duration unreported (datasheet A08240-3); 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 A08240-3); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A08240-3); 8. Primary antibody: Rabbit anti-SF3B3, 2-5 μg/ml (datasheet A08240-3); 9. Primary incubation: Overnight at 4 °C (datasheet A08240-3). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Soft-tissue fibroblasts show low staining (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope differences unreported (UniProt); Regulation: No specific regulator reported (UniProt). Evidence: UniProt Q15393, HPA tissue IHC · Supported, HPA ICC-IF · nucleoplasm, nucleoli and nucleoli rim., Datasheet A08240-3, PMC7999425. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/SF3B3/immunohistochemistry-ihc-guide.