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SMYD3 / Histone-lysine N-methyltransferase SMYD3 · IHC design guide
Design Immunohistochemistry for SMYD3
Plan SMYD3 chromogenic IHC in paraffin sections using the catalog antibody’s documented staining conditions (datasheet PB9893). Compare cytoplasmic staining with the tissue pattern reported by HPA, while allowing for cell cycle dependent nuclear localisation (HPA tissue IHC; UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
SMYD3 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 | Cytoplasmic staining in tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining across tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PB9893) | |
| 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 consistent across sections (standard IHC practice; not target-specific). Selected-image fixative and duration unreported (datasheet PB9893); verify before use. | |
| Caveat | Nuclear accumulation may vary with cell cycle phase (UniProt) | |
| Regulation | Up in colorectal and hepatocellular cancer (UniProt) | |
| Isoform / epitope | 3 isoforms; epitope coverage is unspecified (UniProt; datasheet PB9893) |
Section 1
Recommended SMYD3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol uses EDTA pH 8.0 retrieval (datasheet: PB9893). The published SMYD3 protocols below cover ovarian, endometrial, head and neck, and colorectal specimens (PMC6692630; PMC10774296; PMC11704228; PMC12954928).
| Sample | Paraffin-embedded human intestinal cancer tissue; fixative not specified (datasheet PB9893) |
| Fixation | Image fixative and duration unreported (datasheet PB9893); 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 PB9893); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9893) |
| Primary antibody | Rabbit anti-SMYD3, 0.5-1μg/ml (datasheet PB9893) |
| Primary incubation | Overnight at 4 °C (datasheet PB9893) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9893) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | SMYD3-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in all tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet: PB9893); use each paper’s stated retrieval conditions when reproducing its protocol (PMC10774296; PMC11704228; PMC12954928).
Section 2
What Is the Expected SMYD3 Staining Pattern?
SMYD3 staining in paraffin sections is expected mainly in the cytoplasm, including glandular, respiratory epithelial, hematopoietic and neuronal cells in tissues where HPA reports high signal (HPA tissue IHC). Nuclear staining can also be credible because SMYD3 occupies both compartments and shifts toward the nucleus during S phase and G2/M (UniProt Q9H7B4). SMYD3 has no transmembrane segment (UniProt Q9H7B4 topology). HPA rates its tissue IHC profile Approved, with medium consistency against RNA expression (HPA tissue IHC).
| Clear cytoplasmic signal in colon glandular cells or bronchus respiratory epithelial cells, with discernible tissue structure. | This matches cell types scored High by HPA (HPA tissue IHC). Assess intensity against neighboring cells and controls; high expression does not imply that every cell must stain equally (general IHC practice). |
| Nuclear signal accompanies cytoplasmic signal in a subset of cells. | This can fit SMYD3 biology: it accumulates in the nucleus during S phase and G2/M, while G0/G1-arrested cells are mainly cytoplasmic (UniProt Q9H7B4). Nuclear staining alone is therefore insufficient to call an artefact. |
| A crisp membrane outline, extracellular deposit or stain confined to an implausible compartment dominates the section. | These patterns do not fit SMYD3's reported cytoplasmic and nuclear locations or its lack of a transmembrane segment (UniProt Q9H7B4). Review morphology and detection controls before assigning the signal to SMYD3 (general IHC practice). |
| Staining is concentrated in an unexpected cell population while the HPA-listed cells are weak or unstained. | For example, colon glandular cells are scored High by HPA (HPA tissue IHC). An opposing pattern warrants checks for antibody cross-reactivity, endogenous detection activity and cell identification (general IHC practice); it is not proof of any one cause. |
| Haze covers cells and extracellular spaces, or a tissue scored High shows no interpretable signal. | Diffuse haze prevents compartment scoring (general IHC practice). If colon glandular cells or bone-marrow hematopoietic cells lack signal, first verify the run with a known-positive section (HPA tissue IHC; general IHC practice). |
💡Expected SMYD3 appearanceCall a section positive when identifiable HPA-listed cells show interpretable, mainly cytoplasmic SMYD3 staining, with possible nuclear signal and strong staining in cell types scored High; diffuse haze or a dominant membrane/extracellular pattern is suspect (HPA tissue IHC; UniProt Q9H7B4; general IHC practice).
| Tissue and cell selection | HPA scores colon glandular cells, bronchus respiratory epithelial cells and bone-marrow hematopoietic cells High, while cerebellar granular-layer cells are Low (HPA tissue IHC). Choose controls by cell type; Low is not a validated negative. |
| Cell-cycle-dependent location | SMYD3 is mainly cytoplasmic in G0/G1-arrested cells and accumulates in nuclei during S phase and G2/M (UniProt Q9H7B4). Score cytoplasmic and nuclear signal separately before interpreting a compartment shift. |
| Antibody evidence | HPA lists HPA045821, HPA054352 and CAB012229 as IHC Approved (HPA antibodies). The tissue profile has medium RNA–staining consistency (HPA tissue IHC); agreement with morphology and controls remains useful. |
| Isoforms and processing | UniProt lists three SMYD3 isoforms, no signal peptide or propeptide, and a 1–428 protein chain (UniProt Q9H7B4). Epitope coverage across isoforms is not supplied; do not infer isoform-specific staining or secretion. |
| IF/ICC Q&A: should its distribution match IHC? | HPA reports approved nucleoplasmic and additional cytosolic localisation by ICC-IF, whereas its tissue IHC profile describes cytoplasmic expression (HPA subcellular; HPA tissue IHC). Compare each application with its own evidence. |
| Situation | Likely cause | Next action |
|---|---|---|
| No staining in a colon positive-control section. | An IHC run or antibody step may have failed; colon glandular cells are scored High (HPA tissue IHC; general IHC practice). | Check section identity, antibody application, retrieval, detection reagents and chromogen against the run controls; repeat the run if a control also failed (general IHC practice). |
| Only nuclei stain, with no cytoplasmic signal. | Nuclear SMYD3 is plausible, but a nuclear-only field cannot establish why the distribution differs from HPA tissue IHC (UniProt Q9H7B4; HPA tissue IHC). | Check cell morphology and positive controls, then compare compartments across cells or sections before scoring the pattern (general IHC practice). |
| Membranes or extracellular spaces carry the strongest signal. | This is discordant with reported cytoplasmic and nuclear localisation and the absence of a transmembrane segment (UniProt Q9H7B4). | Inspect detection and no-primary controls for nonspecific or endogenous signal; retain only anatomically interpretable cellular staining (general IHC practice). |
| Diffuse brown background obscures cell boundaries. | Nonspecific reagent binding or endogenous detection activity can obscure chromogenic IHC (general IHC practice). | Review blocking, washes and detection controls; adjust the workflow using the run controls before interpreting intensity (general IHC practice). |
| Unexpected cells stain while HPA-listed glandular cells remain weak. | Cell misidentification, cross-reactivity or endogenous detection activity are possibilities; HPA scores several glandular populations High (HPA tissue IHC; general IHC practice). | Recheck histology and control sections; compare with an independently validated antibody if available (general IHC practice). |
| A Low-scored tissue is unstained and is being treated as a failed run. | HPA lists cerebellar granular-layer cells as Low, not negative, and provides no negative tissue here (HPA tissue IHC). | Judge run performance with a High-scored cell population and technical controls; record the Low-scored result without treating it as a negative standard (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing SMYD3 staining
PB9893 · IHCIHC analysis of SMYD3 using anti-SMYD3 antibody (PB9893). SMYD3 was detected in a paraffin-embedded section of human intestinal cancer tissu
M02080 · IFImmunofluorescent analysis using the Antibody at 1:50 dilution.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000185420-SMYD3/tissue/cerebral+cortex#imid_17257411
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000185420-SMYD3/tissue/colon#imid_17257329
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000185420-SMYD3/tissue/liver#imid_17257346
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000185420-SMYD3/tissue/kidney#imid_17257331
HPA · ICC-IF HEK293Human Protein Atlas immunofluorescence, HEK293, antibody HPA045821; green = SMYD3, blue = nucleus, red = microtubules.
HPA · ICC-IF HEK293Human Protein Atlas immunofluorescence, HEK293, antibody HPA045821; green = SMYD3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for SMYD3 IHC & IF
🧪Run bone marrow first and look for staining in hematopoietic cells (HPA: High in bone marrow hematopoietic cells). HPA detects SMYD3 in all 45 scored tissues, so use no-primary and isotype controls for the negative comparison; cells on the positive slide without visible staining can help assess background, but no specific cell type is established as SMYD3-negative (HPA: no negative tissue rows).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: SMYD3 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 SMYD3 in HEK293, RT-4, U2OS, with annotated localisation: Nucleoplasm (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a host-matched rabbit isotype control, and a SMYD3 knockout specimen or peptide-block control where available (selected-SKU caption: rabbit primary antibody). Quench endogenous peroxidase and check background in bone marrow hematopoietic cells before interpreting chromogenic staining (HPA: High in bone marrow hematopoietic cells; standard IHC practice).
⚠️Feasibility: The selected-SKU paraffin-section caption does not report a fixative, and the supplied evidence reports no SMYD3-specific fixation window or fixation effect (selected-SKU caption: fixative unreported). The caption reports heat retrieval in EDTA at pH 8.0 before IHC detection, but does not establish that retrieval is required (selected-SKU caption: EDTA retrieval). No matched evidence shows that frozen sections or IF are easier than paraffin IHC; HPA ICC-IF images show mainly nucleoplasmic staining with additional cytosolic staining, while endogenous peroxidase can complicate chromogenic interpretation in bone marrow (HPA: subcellular; HPA: High in bone marrow hematopoietic cells; standard IHC practice).
HPA tissue IHC evidence for SMYD3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | 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 → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: SMYD3 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced SMYD3 IHC Tips
Use the PB9893 paraffin-section example as the IHC reference, then assess compartment, tissue morphology and controls when troubleshooting SMYD3 staining (caption PB9893; UniProt Q9H7B4).
Which retrieval condition should I start with for weak SMYD3 staining in paraffin sections?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 (datasheet PB9893). The selected paraffin-section example paired this retrieval with 10% goat serum blocking, 1 μg/ml primary antibody overnight at 4°C, and biotin-based DAB detection (caption PB9893). If signal is weak, compare controlled heating times, such as 10 and 20 min, while keeping cooling, primary concentration and detection constant (standard IHC practice). Test another retrieval condition only as a documented fallback, with the caption’s intestinal cancer section as a reference (caption PB9893; standard IHC practice). Judge any signal gain against tissue integrity and a no-primary control (standard IHC practice).
Can I infer the best fixative for SMYD3 from the published staining pattern?
The selected PB9893 image identifies a paraffin section but does not report its fixative (caption PB9893). Target-specific fixation sensitivity is unknown; the reported tissue staining and SMYD3 topology do not establish a preferred fixative (HPA tissue IHC; UniProt Q9H7B4 topology). Record fixative and exposure duration for each specimen, then compare matched sections using EDTA at pH 8.0 and the 1 μg/ml primary concentration reported for PB9893 (datasheet PB9893; standard IHC practice). Keep detection conditions matched and examine morphology alongside staining intensity (standard IHC practice). If groups differ, assess background and tissue preservation before assigning the difference to epitope masking (standard IHC practice).
How should I evaluate nuclear and cytoplasmic SMYD3 staining in the same section?
Score nuclear and cytoplasmic staining separately in intact cells because SMYD3 is reported in both compartments (UniProt Q9H7B4 subcellular location). Its distribution can shift with cell cycle: mainly cytoplasmic at G0/G1 arrest and more nuclear at S phase and G2/M (UniProt Q9H7B4 subcellular location). HPA reports cytoplasmic tissue staining, while its cell imaging places SMYD3 mainly in the nucleoplasm with additional cytosol staining (HPA tissue IHC; HPA subcellular). Use a counterstain to define nuclei and inspect glandular cells separately from surrounding tissue where relevant (HPA tissue IHC; standard IHC practice). Do not interpret compartment alone as a direct cell-cycle measurement in a static section (standard IHC practice).
Could alternative SMYD3 isoforms explain inconsistent staining between samples?
SMYD3 has 3 reported isoforms, but the supplied PB9893 caption does not identify the antibody epitope or establish isoform coverage (UniProt Q9H7B4 isoforms; caption PB9893). Its annotated SET domain spans residues 4–240, and the protein has no transmembrane segment (UniProt Q9H7B4 domains and topology). Check the antibody’s documented immunogen or epitope against each isoform before attributing a staining difference to splicing (standard IHC practice; UniProt Q9H7B4 isoforms). Compare serial sections under identical EDTA pH 8.0 retrieval and detection conditions to exclude technical variation (datasheet PB9893; standard IHC practice). If epitope information remains unavailable, report isoform selectivity as unresolved (standard IHC practice).
How can IF help resolve which cells and compartments carry SMYD3 signal?
For the separate IF/ICC workflow, multiplex SMYD3 with a marker identifying the cell population being assessed; colon glandular cells are one HPA-reported high-staining population (HPA tissue IHC). Choose a fluorophore channel after checking tissue autofluorescence in an unstained control, and confirm separation with single-stain controls (standard IF practice). Because SMYD3 is reported in the nucleus and cytosol and has no transmembrane segment, assess permeabilisation for access to intracellular epitopes while checking preservation of both compartments (UniProt Q9H7B4 subcellular location and topology; standard IF practice). HPA reports nucleoplasmic signal with additional cytosolic signal in cell imaging, providing a localisation reference (HPA subcellular). Do not transfer PB9893’s chromogenic detection conditions to IF without validation (caption PB9893; standard IF practice).
What should I check when SMYD3 DAB staining appears diffuse or granular?
First compare the stained section with no-primary and detection-only controls to identify signal from the detection system (standard IHC practice). The PB9893 example uses a biotin-based DAB sequence, so include a check for endogenous biotin and a peroxidase block appropriate to the tissue (caption PB9893; standard IHC practice). Inspect folds, cut edges, necrotic areas and pigment before calling granular deposits cellular SMYD3 (standard IHC practice). If background persists, titrate around the caption’s 1 μg/ml primary concentration while holding EDTA retrieval at pH 8.0 and wash conditions constant (caption PB9893; datasheet PB9893; standard IHC practice). Confirm that reduced background preserves interpretable nuclear or cytoplasmic staining (UniProt Q9H7B4 subcellular location; standard IHC practice).
How should I quantify SMYD3 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, and exclude folds, necrosis and section edges (standard IHC practice). Report the percentage of positive cells plus separate nuclear and cytoplasmic intensity scores, or use an H-score for each compartment on a 0–300 scale (standard IHC practice; UniProt Q9H7B4 subcellular location). Normalise cell counts to the number of evaluable cells, or area-based counts to mm² of viable tissue (standard IHC practice). Keep retrieval, DAB development, counterstain and image settings matched across specimens; the PB9893 reference uses EDTA at pH 8.0 (datasheet PB9893; standard IHC practice). Report the scoring threshold and observer or image-analysis method (standard IHC practice).
When is a SMYD3-positive IHC result convincing rather than artefactual?
Look for reproducible cellular staining within intact tissue, with nuclear and/or cytoplasmic localisation consistent with reported SMYD3 distribution (UniProt Q9H7B4 subcellular location; HPA tissue IHC). HPA reports high staining in colon glandular cells, but rates its tissue IHC evidence Approved with medium consistency between staining and RNA expression, so use that pattern as context (HPA tissue IHC). Compare the candidate signal with matched no-primary and detection controls, particularly when using the PB9893 biotin-based DAB sequence (caption PB9893; standard IHC practice). Treat edge staining, necrotic deposits, pigment and endogenous enzyme signal cautiously (standard IHC practice). Document which cell population and compartment support the call (standard IHC practice).
Boster reagents
Best SMYD3 / Histone-lysine N-methyltransferase SMYD3 IHC Antibodies
Anti-SMYD3 antibodies have real IHC data from human paraffin sections (PB9893 IHC caption) and IF images with sample type unreported (M02080 IF captions); catalog reactivity spans human, mouse and rat (catalog: M02080 reactivity).
Real IHC data
Anti-SMYD3 Antibody ®
Cat # PB9893
Real IF data
Anti-SMYD3 Monoclonal Antibody
Cat # M02080
PB9893 has an IHC image from a paraffin section of human intestinal cancer tissue; its listed reactivity is human (PB9893 IHC caption; catalog: PB9893 reactivity). M02080 has IF images without a reported sample type or species, and lists IHC, IF and ICC applications with human, mouse and rat reactivity (M02080 IF captions; catalog: M02080 applications and reactivity).
Which to pick: Choose PB9893 for human paraffin-section IHC because its own caption documents that preparation; the fixative is unreported (PB9893 IHC caption). Choose M02080 for IF/ICC or work across human, mouse and rat because it is monoclonal, lists those applications and species, and has IF images; its IF captions do not identify the samples (catalog: M02080 clone, applications and reactivity; M02080 IF captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9H7B4 (SMYD3_HUMAN, Histone-lysine N-methyltransferase SMYD3).
- Human Protein Atlas. SMYD3 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. SMYD3 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the cytosol..
- Human Protein Atlas. SMYD3 antibody validation summary (3 antibodies).
- Overexpression of SMYD3 in Ovarian Cancer is Associated with Ovarian Cancer Proliferation and Apoptosis via Methylating H3K4 and H4K20. Journal of Cancer 2019 — PMC6692630.
- SMYD3 promotes endometrial cancer through epigenetic regulation of LIG4/XRCC4/XLF complex in non-homologous end joining repair. Oncogenesis 2024 — PMC10774296.
- SMYD3 drives cell cycle and epithelial-mesenchymal transition pathways through dual gene transcriptional repression and activation in HPV-negative head and neck cancer. Scientific reports 2025 — PMC11704228.
- SMYD3 synergises with RACK1 to promote colorectal cancer lung metastasis by recruiting SMAD3. Cell communication and signaling : CCS 2026 — PMC12954928.
- PubMed PMID:15235609 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16710414 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for SMYD3 (Histone-lysine N-methyltransferase SMYD3). Expected localisation Cytoplasmic staining in tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PB9893). Validated IHC antibody: PB9893 at 0.5-1μg/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 intestinal cancer tissue; fixative not specified (datasheet PB9893); 2. Fixation: Image fixative and duration unreported (datasheet PB9893); 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 PB9893); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9893); 8. Primary antibody: Rabbit anti-SMYD3, 0.5-1μg/ml (datasheet PB9893); 9. Primary incubation: Overnight at 4 °C (datasheet PB9893). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific). Selected-image fixative and duration unreported (datasheet PB9893); verify before use.; Caveat: Nuclear accumulation may vary with cell cycle phase (UniProt); Isoform / epitope: 3 isoforms; epitope coverage is unspecified (UniProt; datasheet PB9893); Regulation: Up in colorectal and hepatocellular cancer (UniProt). Evidence: UniProt Q9H7B4, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet PB9893, PMC6692630, PMC10774296. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/SMYD3/immunohistochemistry-ihc-guide.