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SETD3 / Actin-histidine N-methyltransferase · IHC design guide
Design Immunohistochemistry for SETD3
Plan SETD3 staining in paraffin sections around cytoplasmic expression in most tissues and high staining in several glandular cell populations (HPA tissue IHC). The catalog antibody has an IHC dilution of 2–5 μg/ml (datasheet A10965-1); use consistent fixation and appropriate controls (standard IHC practice).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
SETD3 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 tissue staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in most tissues; high in several glandular cell populations (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A10965-1) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Expression regulation not annotated (UniProt) | |
| Isoform / epitope | 3 isoforms; epitope effects undetermined (UniProt) |
Section 1
Recommended SETD3 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet A10965-1) with the published human tissue microarray and mouse liver protocol (PMC9329778).
| Sample | Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A10965-1) |
| Fixation | Image fixative and duration unreported (datasheet A10965-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A10965-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A10965-1) |
| Primary antibody | Rabbit anti-SETD3, 2-5 μg/ml (datasheet A10965-1) |
| Primary incubation | Overnight at 4 °C (datasheet A10965-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A10965-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | SETD3-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet A10965-1); the published protocol does not specify retrieval (PMC9329778).
Section 2
What Is the Expected SETD3 Staining Pattern?
In paraffin-section IHC, expect predominantly cytoplasmic SETD3 staining in glandular cells of tissues such as colon and duodenum (HPA: cytoplasmic expression in most tissues; High in these glandular cells). UniProt places SETD3 mainly in the cytoplasm, also in the nucleus, and reports no transmembrane segment (UniProt Q86TU7: localization and topology). Treat tissue predictions as guides: HPA rates its tissue IHC profile Approved, with medium RNA–staining consistency and external verification pending.
| Colon or duodenum glandular cells show clear cytoplasmic chromogen, with recognizable cell boundaries and a clean surrounding field. | This fits the expected IHC distribution (HPA: High in colon and duodenum glandular cells; cytoplasmic expression in most tissues). Compare intensity within the same staining run; HPA’s High category does not prescribe a numerical score or a particular chromogen shade. |
| Signal is confined to nuclei, membranes, or another compartment without discernible cytoplasmic staining. | Question the IHC result and check staining controls and morphology (UniProt Q86TU7: mainly cytoplasmic; HPA: cytoplasmic tissue IHC). Some nuclear localization is recorded by UniProt, so a nuclear component alone does not invalidate an otherwise convincing cytoplasmic pattern. |
| Adipocytes or skeletal myocytes stain prominently while expected glandular cells do not. | Consider antibody cross-reactivity or endogenous detection activity, then review controls and cell identification (HPA: Not detected in adipocytes and skeletal myocytes; High in colon glandular cells). These are cell-level reference observations, not a claim that every cell in those tissues must be negative. |
| Brown signal spreads across stroma, empty spaces, or most cells without clear cellular boundaries. | Treat this as diffuse background until controls show otherwise. In chromogenic IHC, incomplete blocking, residual detection activity, or excessive reagent concentration can obscure cell-level interpretation (general IHC practice). HPA’s cytoplasmic tissue profile does not establish that diffuse staining is SETD3. |
| A colon or duodenum section has no glandular-cell signal, yet morphology appears intact. | Call the run inconclusive until a known-positive control and detection controls are reviewed (HPA: High in colon and duodenum glandular cells). Absence of staining can reflect a failed IHC workflow; this evidence does not identify SETD3-specific fixation sensitivity. |
💡Expected SETD3 appearanceA convincing positive is distinct, predominantly cytoplasmic chromogen in glandular cells of a reference tissue such as colon or duodenum (HPA: High in these cells; cytoplasmic tissue IHC); widespread cell-independent haze or isolated staining of HPA-undetected cell types is suspect (HPA: Not detected in adipocytes and skeletal myocytes).
| Reference tissue and cell type | Select a section containing identifiable glandular cells for a positive control (HPA: High in appendix, colon, duodenum, rectum, small intestine, and stomach glandular cells). Score the named cells, since a tissue label alone does not define every cell’s SETD3 status. |
| Compartment and topology | Prioritize cytoplasmic staining in tissue IHC (HPA: cytoplasmic expression in most tissues; UniProt Q86TU7: mainly cytoplasmic). UniProt also lists the nucleus and no transmembrane segment; it does not support a membrane-only IHC pattern. |
| Strength of tissue evidence | Use HPA levels as reference observations, not universal intensity thresholds (HPA: tissue IHC Approved; medium consistency with RNA; external verification pending). The listed Not detected findings apply to specified cells, including adipocytes and smooth muscle cells (HPA: tissue IHC). |
| Antibody validation and molecular forms | Two listed antibodies have Approved IHC status (HPA: HPA003591 and HPA003639). UniProt lists three isoforms and a phosphoserine at residue 513 (UniProt Q86TU7); the supplied evidence does not identify the antibody epitope or show isoform-dependent staining. |
| IF/ICC Q&A: should mitochondrial signal match tissue IHC? | HPA reports mainly mitochondrial localization in ICC-IF and an Approved ICC result for HPA003591 (HPA: subcellular ICC-IF; antibody status). Interpret that assay separately from HPA’s predominantly cytoplasmic tissue IHC profile; neither record establishes an IF protocol here. |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in glandular cells of a reference section. | The staining or detection run may have failed; absence alone cannot identify the failed step (HPA: High in colon and duodenum glandular cells). | Check a known-positive control, section integrity, reagent preparation, and detection steps; review retrieval against the validated IHC workflow (general IHC practice). Do not infer SETD3-specific fixation sensitivity from HPA staining levels. |
| Signal is faint in a reference tissue. | Low contrast may reflect assay conditions, while HPA’s High category is a cell-level reference rather than a guaranteed intensity in every run (HPA: High in colon glandular cells). | Compare the control section and detection controls from the same run; confirm antibody dilution and chromogen development against the validated IHC workflow (general IHC practice). |
| The entire section has diffuse brown background. | Nonspecific binding or residual endogenous detection activity can produce widespread chromogen (general chromogenic IHC practice). | Review the no-primary control, blocking, washes, and endogenous-activity controls appropriate to the detection chemistry (general IHC practice). Interpret SETD3 only where cellular staining remains distinct. |
| Nuclei or cell borders dominate the signal. | A compartment mismatch raises concern about specificity or interpretation (HPA: cytoplasmic tissue IHC; UniProt Q86TU7: mainly cytoplasmic, also nuclear). | Verify cell boundaries with the counterstain, compare a reference section, and review controls. Do not reject a modest nuclear component solely because it is nuclear (UniProt Q86TU7: nucleus listed). |
| Adipocytes or smooth muscle cells stain more strongly than reference glandular cells. | Possible cross-reactivity or detection background requires checking; HPA reports those specific cell types as Not detected (HPA: adipocytes and smooth muscle cells). | Confirm cell identity and examine the no-primary and positive controls (general IHC practice). Record staining by cell type rather than assigning a whole tissue a positive or negative result. |
| Tissue IHC appears cytoplasmic, but an ICC-IF image appears mitochondrial. | The supplied assay observations differ (HPA: cytoplasmic tissue IHC; mainly mitochondrial ICC-IF); they do not establish which preparation step accounts for the difference. | Report each result with its assay and controls. Evaluate paraffin-section staining against the tissue IHC reference, and use the separate IF/ICC guide for that assay. |
Experiment evidence
IHC & IF images showing SETD3 staining
A10965-1 · IHCIHC analysis of SETD3 using anti-SETD3 antibody (A10965-1). SETD3 was detected in a paraffin-embedded section of human breast cancer tissue.
A10965-1 · IHCIHC analysis of SETD3 using anti-SETD3 antibody (A10965-1). SETD3 was detected in a paraffin-embedded section of human liver cancer tissue.
A10965-1 · IFIF analysis of SETD3 using anti-SETD3 antibody (A10965-1). SETD3 was detected in an immunocytochemical section of U20S cells. Enzyme antigen
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000183576-SETD3/tissue/cerebral+cortex#imid_1346564
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000183576-SETD3/tissue/colon#imid_1346490
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000183576-SETD3/tissue/liver#imid_1346483
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000183576-SETD3/tissue/kidney#imid_1346484
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA003591; green = SETD3, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA003591; green = SETD3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for SETD3 IHC & IF
🧪Run colon first: glandular cells should stain (HPA: Colon glandular cells High). Use adipose tissue adipocytes as the negative comparator (HPA: Adipocytes Not detected); on the colon slide, assess non-glandular cells as internal negative candidates, but do not assume they are SETD3-negative because the supplied HPA row rates only glandular cells (HPA: Colon glandular cells High).
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show SETD3 in A-431, U-251MG, U2OS, with annotated localisation: Mitochondria (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a concentration-matched nonimmune rabbit IgG isotype control (caption: rabbit anti-SETD3 primary); use a SETD3 knockout specimen or antigen-peptide block as a biological specificity control if available (standard IHC practice). Quench endogenous peroxidase and check background in colon inflammatory cells before interpreting DAB staining (standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A10965-1 paraffin-section caption does not state the fixative (selected-SKU caption: fixative not stated). The reported IHC example uses heat retrieval in EDTA at pH 8.0 before chromogenic detection, so retrieval is a practical starting condition rather than an established requirement for every specimen (selected-SKU caption: EDTA pH 8.0, DAB). IF/ICC Q&A—Is it easier than paraffin IHC? The supplied evidence does not establish that; HPA has ICC-IF images and reports mainly mitochondrial localisation, whereas UniProt reports mainly cytoplasmic localisation, so assess localisation against controls in the chosen preparation (HPA: subcellular summary; UniProt: subcellular location).
HPA tissue IHC evidence for SETD3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Pending external verification.). 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 |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | High | Protein (IHC) | HPA → |
| Rectum | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Ovary | Follicle cells | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced SETD3 IHC Tips
Use matched paraffin sections and compartment-aware controls to troubleshoot SETD3 staining; tissue IHC and cell IF report different localisation patterns (HPA tissue IHC; HPA subcellular).
How should I recover SETD3 staining when paraffin sections are weak?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A10965-1). The selected image used this retrieval before 2 μg/ml primary antibody overnight at 4°C; its caption gives no retrieval time (datasheet A10965-1). Keep heating, cooling and section thickness consistent across a positive control and test sections so differences reflect the sample more reliably (standard IHC practice). If staining remains weak, compare a citrate buffer at pH 6.0 on serial sections as a fallback, documenting any change in background (standard IHC practice). Score glandular-cell cytoplasm in colon or appendix as a reference compartment (HPA: high in colon and appendix glandular cells; HPA: cytoplasmic expression in most tissues).
Could fixation explain variable SETD3 staining between paraffin blocks?
Target-specific sensitivity to fixation is unknown: the selected paraffin-section caption does not state a fixative (datasheet A10965-1). Record the fixative, time before fixation, duration and processing schedule for each block, then compare sections retrieved together in EDTA at pH 8.0 (standard IHC practice; datasheet A10965-1). Include a consistently processed positive control, such as colon glandular cells, in every run (HPA: high in colon glandular cells; standard IHC practice). If blocks differ, inspect nuclear detail and tissue preservation before changing antibody concentration; damaged morphology can confound compartment scoring (standard IHC practice). Do not attribute fixation sensitivity to SETD3’s localisation or phosphorylation without direct fixation evidence (UniProt Q86TU7: mainly cytoplasmic, phosphoserine at residue 513).
Should SETD3 staining be cytoplasmic, nuclear or punctate?
For tissue IHC, expect predominantly cytoplasmic staining across many tissues, while allowing for reported nuclear localisation (HPA tissue IHC: cytoplasmic expression in most tissues; UniProt Q86TU7: cytoplasm and nucleus, mainly cytoplasmic). Score cytoplasm and nuclei separately in the same defined cell population, using matched exposure and DAB development conditions (standard IHC practice). A punctate pattern warrants comparison with serial sections and the no-primary control before assigning it to organelles (standard IHC practice). HPA cell IF reports mitochondria as its approved main location, which creates a compartment difference to document rather than resolve by appearance alone (HPA subcellular: mitochondria approved). Keep the 2 assay types distinct in the report (HPA tissue IHC; HPA subcellular).
How could isoforms or epitope accessibility change the IHC pattern?
SETD3 has 3 reported isoforms and a SET domain spanning residues 94–314, but the supplied caption does not identify this antibody’s epitope (UniProt Q86TU7; datasheet A10965-1). Check the antibody’s stated immunogen or mapped epitope before claiming that staining represents every isoform (standard antibody validation practice). Compare serial sections under the same EDTA pH 8.0 retrieval and primary concentration, then assess whether altered retrieval changes cell staining or diffuse background (datasheet A10965-1; standard IHC practice). SETD3 also has reported phosphorylation at residue 513; no supplied evidence links that modification to antibody binding (UniProt Q86TU7; datasheet A10965-1). Treat compartment differences as observations until epitope coverage and specificity are verified (standard IHC practice).
How can IF help investigate a disputed SETD3 IHC compartment?
Use IF as a separate compartment check: tissue IHC is broadly cytoplasmic, whereas HPA cell IF assigns SETD3 mainly to mitochondria (HPA tissue IHC; HPA subcellular). Multiplex with a validated mitochondrial marker and a nuclear counterstain, then inspect signal overlap within individual cells rather than equating neighboring signals (HPA subcellular: mitochondria approved; standard IF practice). Choose a fluorophore in a relatively low-autofluorescence channel for the specimen and include single-label and no-primary controls (standard IF practice). SETD3 has no transmembrane segment, but this antibody’s epitope location is unspecified; test gentle plasma-membrane permeabilisation and avoid assuming access to a mitochondrial interior epitope (UniProt Q86TU7 topology; datasheet A10965-1; standard IF practice).
What should I change when DAB obscures SETD3-positive cells?
First inspect a no-primary section and a secondary-only section for staining unrelated to the SETD3 primary (standard IHC practice). The selected paraffin-section image used 10% goat serum, 2 μg/ml primary overnight at 4°C, and DAB detection (datasheet A10965-1). Block endogenous peroxidase before HRP detection, keep DAB development time identical across sections, and check whether signal follows tissue edges or damaged areas (standard chromogenic IHC practice). If diffuse staining persists, titrate primary antibody below the documented concentration while retaining a known positive section (datasheet A10965-1; standard IHC practice). Judge specificity in glandular-cell cytoplasm, with colon as one supplied high-expression reference (HPA: high in colon glandular cells; HPA tissue IHC: cytoplasmic expression).
How should I quantify SETD3 across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring; HPA reports cytoplasmic staining in most tissues and high glandular-cell staining in colon (HPA tissue IHC). For comparable regions, report the percentage of positive cells and an H-score from 0–300, using intensity categories 0–3 with fixed thresholds (standard IHC scoring practice). If cell density varies, add positive cells per mm² and normalise to the number or area of eligible, intact cells, excluding necrosis and section folds (standard histology practice). Process and image all sections with the same EDTA pH 8.0 retrieval and DAB endpoint (datasheet A10965-1; standard IHC practice). Report cytoplasmic and nuclear scores separately because both locations are annotated (UniProt Q86TU7).
When is apparent SETD3 positivity more likely to be artefact?
A credible tissue result shows reproducible intracellular staining in the expected cell population, with cytoplasm assessed first (HPA tissue IHC: cytoplasmic expression in most tissues; standard IHC practice). Compare high-staining colon glandular cells with a supplied low or undetected reference, such as thyroid glandular cells or adipocytes, while recognising HPA’s medium RNA agreement and pending external verification (HPA tissue IHC: colon high, thyroid low, adipocytes not detected; HPA reliability description). Treat signal limited to section edges, necrosis or the no-primary control as suspect (standard IHC practice). Isolated nuclear or mitochondrial-looking DAB deposits need corroboration before compartment assignment, given the differing tissue IHC, UniProt and cell IF annotations (HPA tissue IHC; UniProt Q86TU7; HPA subcellular). Check endogenous peroxidase blocking when control sections develop DAB (standard chromogenic IHC practice).
Boster reagents
Best SETD3 / Actin-histidine N-methyltransferase IHC Antibodies
A10965-1 has IHC images from human paraffin-embedded cancer sections and IF images from human breast cancer tissue and U20S cells (catalog image captions); catalog reactivity lists human, mouse and rat (catalog reactivity).
Real IHC data
Anti-SETD3 Antibody ®
Cat # A10965-1
A10965-1 has chromogenic IHC images from paraffin-embedded human breast, liver, ovarian serous and renal cancer sections (IHC image captions). A10965-1 also lists IF and ICC applications, with IF images from U20S cells and paraffin-embedded human breast cancer tissue (catalog applications; IF image captions).
Which to pick: For chromogenic tissue IHC, choose A10965-1: its IHC captions document paraffin sections, EDTA retrieval at pH 8.0, and 2 μg/ml primary antibody with DAB detection; the fixative is unreported (IHC image captions). For IF/ICC, A10965-1 lists both applications and has IF images from U20S cells and human breast cancer tissue at 5 μg/ml (catalog applications; IF image captions). For mouse or rat work, A10965-1 lists species reactivity, but its supplied IHC and IF images show human samples only; it is rabbit hosted, and clonality is unreported (catalog reactivity and host; IHC and IF image captions; catalog clone field).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q86TU7 (SETD3_HUMAN, Actin-histidine N-methyltransferase).
- Human Protein Atlas. SETD3 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. SETD3 subcellular location (ICC-IF): Mainly localized to the mitochondria..
- Human Protein Atlas. SETD3 antibody validation summary (2 antibodies).
- Stabilization of SETD3 by deubiquitinase USP27 enhances cell proliferation and hepatocellular carcinoma progression. Cellular and molecular life sciences : CMLS 2022 — PMC8752572.
- SETD3 Methyltransferase Regulates PLK1 Expression to Promote In Situ Hepatic Carcinogenesis. Frontiers in oncology 2022 — PMC9329778.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:12508121 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for SETD3 (Actin-histidine N-methyltransferase). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A10965-1). Validated IHC antibody: A10965-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A10965-1); 2. Fixation: Image fixative and duration unreported (datasheet A10965-1); 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: EDTA pH 8.0 (datasheet A10965-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A10965-1); 8. Primary antibody: Rabbit anti-SETD3, 2-5 μg/ml (datasheet A10965-1); 9. Primary incubation: Overnight at 4 °C (datasheet A10965-1). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope effects undetermined (UniProt); Regulation: Expression regulation not annotated (UniProt). Evidence: UniProt Q86TU7, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the mitochondria., Datasheet A10965-1, PMC9329778. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/SETD3/immunohistochemistry-ihc-guide.