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NSUN6 / tRNA (cytosine(72)-C(5))-methyltransferase NSUN6 · IHC design guide
Design Immunohistochemistry for NSUN6
Plan NSUN6 chromogenic IHC around granular cytoplasmic staining, with strong signal reported in ciliated cell bodies (HPA tissue IHC). Compare these cells with HPA tissue-IHC cell populations where staining was not detected, and start the catalog antibody at 2–5 μg/ml (HPA tissue IHC; datasheet A13404-3).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NSUN6 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 | Granular cytoplasm in most tissues (HPA tissue IHC) | |
| Staining pattern | Strong granular cytoplasmic staining in ciliated cell bodies (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A13404-3) | |
| Positive control | Bronchus+4 more · see all | |
| Negative control | Adipose tissue+3 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific) | |
| Caveat | Staining has medium consistency with RNA data (HPA tissue IHC) | |
| Regulation | No expression regulator annotated (UniProt) | |
| Isoform / epitope | No isoforms annotated; one 1–469 chain (UniProt) |
Section 1
Recommended NSUN6 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: A13404-3) is accompanied by published NSUN6 IHC methods for ovarian, renal, and cervical cancer samples (PMC13454582; PMC13270792; PMC11225205).
| Sample | Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet A13404-3) |
| Fixation | Image fixative and duration unreported (datasheet A13404-3); 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 A13404-3); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A13404-3) |
| Primary antibody | Rabbit anti-NSUN6, 2-5 μg/ml (datasheet A13404-3) |
| Primary incubation | Overnight at 4 °C (datasheet A13404-3) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A13404-3) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | NSUN6-positive staining in ciliated cells (cell body) of bronchus (HPA tissue IHC: High). HPA tissue profile: Granular cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet: A13404-3); citrate retrieval for 3 min is a published alternative (PMC13454582).
Section 2
What Is the Expected NSUN6 Staining Pattern?
In paraffin-section IHC, expect granular cytoplasmic NSUN6 staining in most tissues, with high staining in the cell bodies of ciliated cells in bronchus, fallopian tube, and nasopharynx (HPA: tissue IHC). NSUN6 is cytoplasmic and has no transmembrane segment (UniProt Q8TEA1). HPA rates its tissue IHC profile Approved, with medium consistency between staining and RNA data; external verification remains pending (HPA: tissue IHC reliability).
| Granular cytoplasmic staining is high in bronchial, fallopian tube, or nasopharyngeal ciliated cell bodies. | This matches the reported location and strongest listed cell-level examples (HPA: granular cytoplasmic profile; High in ciliated cells). Judge staining in the named cells, not from a whole-section average: neighboring cell types need not show the same intensity. |
| Staining is predominantly nuclear or confined to cell borders, without the expected cytoplasmic granules. | That distribution conflicts with the paraffin-section profile and cytoplasmic annotation (HPA: tissue IHC profile; UniProt Q8TEA1 subcellular location). Treat an isolated compartment mismatch as suspect; review morphology, the negative control, and a known-positive section before assigning it to NSUN6. |
| Strong staining appears in adipocytes, ovarian stroma, smooth muscle cells, or splenic red-pulp cells. | Those specific cells were reported as not detected in the listed tissues (HPA: tissue IHC negatives). Consider cross-reactivity or endogenous chromogenic detection activity, both general IHC possibilities; compare a reagent control and inspect whether pigment or staining follows cell boundaries. |
| Diffuse color covers cells and extracellular spaces without a clear granular cytoplasmic pattern. | This is difficult to score as NSUN6 because HPA describes granular cytoplasmic expression (HPA: tissue IHC profile). General IHC causes include nonspecific reagent binding, residual detection activity, or precipitated chromogen; assess a negative control and examine whether color tracks tissue structures. |
| No staining is visible in the ciliated cell bodies of a bronchus positive-control section. | This disagrees with the reported High staining for those cells (HPA: High in bronchial ciliated cells). First confirm that the section contains intact ciliated cells, then check the run controls and reagent steps. HPA's Approved rating still carries pending external verification (HPA: tissue IHC reliability). |
💡Expected NSUN6 appearanceCall a positive result when high granular cytoplasmic color marks ciliated cell bodies in bronchus, fallopian tube, or nasopharynx (HPA: tissue IHC); dominant nuclear staining or strong signal in HPA-listed undetected cells is suspect (HPA: tissue IHC profile and negatives).
| Cell-level positive controls | Bronchus, fallopian tube, and nasopharynx have High staining specifically in ciliated cell bodies (HPA: tissue IHC). Use the relevant cells as the reference; a tissue name alone does not identify which cells should stain. |
| Cell-level negative and lower-signal comparisons | Adipocytes and smooth muscle cells are listed as not detected, while cerebral-cortex neurons and lung alveolar cells are Low (HPA: tissue IHC). Low is a weaker expected observation, not a negative-control designation. |
| Evidence strength | The tissue pattern is Approved but has medium staining-versus-RNA consistency and awaits external verification (HPA: tissue IHC reliability). One antibody, HPA045902, is listed as IHC Approved (HPA: antibody validation); do not treat the pattern as independently confirmed. |
| Topology and processing | NSUN6 has no transmembrane segment, signal peptide, propeptide, or annotated isoforms; its recorded chain spans residues 1–469 (UniProt Q8TEA1). These facts support intracellular interpretation but do not establish an epitope location or a target-specific retrieval requirement. |
| IF/ICC question: should its pattern match paraffin-section IHC? | HPA reports an approved Golgi apparatus location by ICC-IF, while tissue IHC describes granular cytoplasm (HPA: subcellular ICC-IF; HPA: tissue IHC). Interpret each assay against its own reported pattern; this section supplies no IF/ICC protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| The bronchus positive control is blank. | The expected ciliated cells may be absent from the plane of section, or the staining run may have failed (HPA: High in bronchial ciliated cells; general IHC practice). | Confirm ciliated-cell morphology, inspect a run control, then verify antibody application, detection reagents, and chromogen development (general IHC practice). Avoid calling the test tissue NSUN6-negative until the control works. |
| Color is mainly nuclear, membranous, or extracellular. | This disagrees with the granular cytoplasmic tissue pattern (HPA: tissue IHC) and cytoplasmic annotation (UniProt Q8TEA1). | Recheck compartment assignment at higher magnification; compare the positive tissue and a negative reagent control. If the mismatch persists, report it as discordant staining rather than a confirmed NSUN6 distribution (general IHC practice). |
| Listed negative cell types stain strongly. | Strong color in adipocytes, ovarian stroma, smooth muscle cells, or splenic red-pulp cells conflicts with their not-detected entries (HPA: tissue IHC negatives). Cross-reactivity or endogenous activity is possible (general IHC practice). | Compare a negative reagent control and inspect the distribution of color. Where the detection system permits, check its endogenous-activity control before interpreting strong staining as target expression (general IHC practice). |
| Background is diffuse across the section. | A diffuse film does not resemble the reported granules (HPA: tissue IHC profile); nonspecific binding or chromogen deposit can obscure cells (general IHC practice). | Check negative controls, wash and blocking steps, and chromogen deposition. Score only distinguishable cellular staining against the background (general IHC practice). |
| A low-signal tissue appears nearly blank. | HPA lists cerebral-cortex neurons, lung alveolar cells, and several other cell types as Low, so weak visibility can be consistent with the reference profile (HPA: tissue IHC low entries). | Use a High ciliated-cell reference to assess run performance, then document the low-signal tissue at cell level. Do not promote a weak reference into a definitive negative control. |
| IHC appears granular, but an ICC-IF image emphasizes Golgi staining. | The observations come from different assay contexts: granular cytoplasm in tissue IHC and approved Golgi localization in ICC-IF (HPA: tissue IHC; HPA: subcellular ICC-IF). | Compare each image with the HPA pattern for its own assay. For this paraffin-section IHC decision, prioritize cell-level tissue staining and its controls (HPA: tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing NSUN6 staining
A13404-3 · IHCIHC analysis of NSUN6 using anti-NSUN6 antibody (A13404-3). NSUN6 was detected in a paraffin-embedded section of human liver cancer tissue.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000241058-NSUN6/tissue/cerebral+cortex#imid_12722445
HPA · StomachHuman Protein Atlas IHC, Stomach. Source: https://www.proteinatlas.org/ENSG00000241058-NSUN6/tissue/stomach+1#imid_12722329
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000241058-NSUN6/tissue/colon#imid_12722363
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000241058-NSUN6/tissue/liver#imid_12722372
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA045902; green = NSUN6, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA045902; green = NSUN6, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NSUN6 IHC & IF
🧪Run bronchus first and score strong staining in ciliated cell bodies (HPA: High in bronchial ciliated cells). Use adipose tissue as the negative tissue and assess adipocytes for absent staining (HPA: Not detected in adipocytes); on the bronchus slide, compare adjacent nonciliated cells with the ciliated cells without assuming they are NSUN6-negative (HPA: High is specified for ciliated cells).
Positive control tissue: Bronchus (Ciliated cells (cell body), HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show NSUN6 in A-431, U-251MG, U2OS, with annotated localisation: Golgi apparatus (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a concentration-matched rabbit isotype control matched to the catalog antibody’s clonality, and a validated NSUN6 knockout specimen as a biological negative (selected-SKU caption: rabbit primary antibody; standard IHC practice). Quench endogenous peroxidase and check bronchial mucus or debris for background DAB signal (selected-SKU caption: peroxidase/DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU tissue-IHC caption does not state the fixative (selected-SKU caption: fixative unreported). Paraffin-section IHC is documented with heat-mediated EDTA retrieval at pH 8.0, but the caption does not establish that retrieval is required (selected-SKU caption: EDTA retrieval, pH 8.0). Frozen sections are not established as easier; ICC-IF images exist, while bronchial mucus or debris warrants a background check during chromogenic scoring (HPA: ICC-IF images in A-431, U-251MG and U2OS; standard IHC practice).
HPA tissue IHC evidence for NSUN6
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 |
|---|---|---|---|---|
| Bronchus | Ciliated cells (cell body) | High | Protein (IHC) | HPA → |
| Fallopian tube | Ciliated cells (cell body) | High | Protein (IHC) | HPA → |
| Nasopharynx | Ciliated cells (cell body) | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Ovary | Ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NSUN6 IHC Tips
Use the catalog antibody’s paraffin-section result as a starting point, then assess staining by cell type, compartment and control performance.
Which retrieval conditions should I start with for NSUN6 in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 (datasheet A13404-3). The selected paraffin-section image used this condition before incubation with 2 μg/ml primary antibody overnight at 4°C (datasheet A13404-3). If staining is weak, compare retrieval duration on matched sections while keeping antibody concentration and detection constant (standard IHC practice). Assess tissue preservation alongside signal, because excessive heating can damage morphology and make granular cytoplasmic staining difficult to score (standard IHC practice; HPA: granular cytoplasmic expression). Record the heating method and duration for each run so a stronger signal can be reproduced (standard IHC practice).
How should I troubleshoot fixation-related loss of NSUN6 staining?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative (datasheet A13404-3). Record the fixative and fixation interval for each specimen, and compare matched material when poor staining tracks with processing history (standard IHC practice). Keep EDTA retrieval at pH 8.0 as the starting condition while changing one processing variable at a time (datasheet A13404-3; standard IHC practice). Inspect nuclear detail and tissue integrity before attributing a weak signal to antigen loss (standard IHC practice). The HPA staining profile cannot establish which fixative preserves this antibody’s epitope (HPA: tissue IHC profile; datasheet A13404-3: fixative unreported).
Should NSUN6 staining be cytoplasmic or concentrated near the Golgi?
Expect mainly granular cytoplasmic staining when reading tissue sections (HPA: granular cytoplasmic expression in most tissues). UniProt also assigns NSUN6 to the cytoplasm, while HPA reports an approved Golgi apparatus location in cell imaging (UniProt Q8TEA1: cytoplasm; HPA: approved Golgi apparatus location). Compare candidate signal with nearby negative cells and with the section’s morphology before calling a compact perinuclear pattern positive (standard IHC practice). A predominantly nuclear or surface-restricted signal warrants scrutiny because it does not match those reported locations (UniProt Q8TEA1: cytoplasm; HPA: Golgi apparatus). Use compartment and cell-type consistency together; location alone does not establish antibody specificity (standard IHC practice).
Could an NSUN6 isoform or inaccessible epitope explain inconsistent staining?
The supplied record lists 0 isoforms and one 1–469 chain, so an annotated isoform difference does not explain discordant sections (UniProt Q8TEA1: isoforms and processing). NSUN6 has a PUA domain at residues 111–203 and acetyllysine reported at residue 419, but the antibody epitope is unspecified (UniProt Q8TEA1: domains and modified residues; datasheet A13404-3: epitope unreported). Do not assign an epitope to either region from these annotations (standard IHC interpretation). Compare matched sections under the stated EDTA pH 8.0 retrieval and consistent detection before invoking epitope masking (datasheet A13404-3; standard IHC practice). Seek independent antibody or genetic validation if the discrepancy persists (standard IHC practice).
How can I assess NSUN6 by IF alongside a cell-type marker?
Treat IF as a separately optimised application; the selected antibody evidence describes chromogenic paraffin-section IHC (datasheet A13404-3). For ciliated epithelium, multiplex with a validated ciliated-cell marker and compare NSUN6 signal within marker-positive and adjacent cells (HPA: high staining in bronchus, fallopian tube and nasopharynx ciliated cells; standard IF practice). Choose fluorophores after checking tissue autofluorescence in unstained and secondary-only controls, and separate emission channels adequately (standard IF practice). Because NSUN6 has no transmembrane segment and is reported intracellularly, permeabilise fixed cells to expose intracellular epitopes, while optimising detergent strength for morphology (UniProt Q8TEA1: topology and cytoplasm; standard IF practice). A Golgi-enriched IF pattern can be compared with HPA imaging without assuming identical tissue-IHC appearance (HPA: approved Golgi apparatus location; HPA: granular cytoplasmic tissue staining).
What should I check when NSUN6 DAB staining is diffuse or widespread?
First compare primary-omission and secondary-only controls with the stained section to locate nonspecific detection (standard IHC practice). The selected caption used 10% goat serum blocking, a peroxidase-conjugated secondary and DAB development after 2 μg/ml primary incubation (datasheet A13404-3). Check endogenous peroxidase quenching and shorten chromogen development if control sections accumulate brown precipitate (standard chromogenic IHC practice). Distinguish cellular granules from pigment, folds, edge staining and diffuse extracellular deposits on matched morphology (standard IHC practice; HPA: granular cytoplasmic expression). Background across cells reported as unstained, such as adipocytes, should prompt a fresh review of blocking, detection and exposure (HPA: adipocytes not detected; standard IHC practice).
How should I quantify NSUN6 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the anatomical region and cell class before scoring, because HPA reports different staining across cell types (HPA: tissue IHC profile). For chromogenic sections, record the percentage of positive cells and staining intensity, or calculate an H-score from intensity categories 0–3 for a possible range of 0–300 (standard IHC scoring practice). Normalise counts to eligible nucleated cells or sampled tissue area, and report sampling area and exclusion rules (standard IHC practice). Score cytoplasmic signal separately from nuclear or extracellular color, using one threshold across comparably processed sections (HPA: granular cytoplasmic expression; standard IHC practice). Include control performance and processing batch when comparing groups (standard IHC practice).
How can I distinguish credible NSUN6 signal from tissue artefact?
A credible pattern should show cellular, granular cytoplasmic staining in plausible cell populations, with control sections behaving as expected (HPA: granular cytoplasmic expression; standard IHC practice). High staining in ciliated cells provides a useful positive comparison, while adipocytes and smooth muscle cells are reported as unstained in the HPA tissue survey (HPA: ciliated cells high; HPA: adipocytes and smooth muscle cells not detected). Question staining confined to section edges, folds, necrotic regions or tissue surfaces, and check endogenous peroxidase with appropriate controls (standard IHC practice). Predominantly nuclear staining needs independent confirmation against the cytoplasmic and Golgi location reports (UniProt Q8TEA1: cytoplasm; HPA: Golgi apparatus). HPA labels its tissue staining approved but notes medium RNA agreement and pending external verification, so orthogonal validation remains useful (HPA: reliability assessment).
Boster reagents
Best NSUN6 / tRNA (cytosine(72)-C(5))-methyltransferase NSUN6 IHC Antibodies
A13404-3 has an IHC image from a paraffin-embedded human liver cancer section (catalog image caption). Human, Mouse, and Rat reactivity is listed (catalog reactivity); no IF image is supplied (catalog IF images).
Real IHC data
Anti-NSUN6 Antibody ®
Cat # A13404-3
A13404-3 is listed for IHC and for Human, Mouse, and Rat reactivity (catalog applications/reactivity). Its IHC image documents a paraffin-embedded human liver cancer section, EDTA retrieval at pH 8.0, and 2 μg/ml primary antibody (A13404-3 image caption).
Which to pick: Choose A13404-3 for chromogenic IHC on paraffin sections: its own caption documents DAB detection in human liver cancer tissue; the fixative is unreported (A13404-3 image caption). No IF/ICC-validated SKU is supplied, and clonality is unreported (catalog applications, IF images, and clone field). For cross-species planning, A13404-3 lists Human, Mouse, and Rat reactivity, but its IHC image documents human tissue only (catalog reactivity; A13404-3 image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8TEA1 (NSUN6_HUMAN, tRNA (cytosine(72)-C(5))-methyltransferase NSUN6).
- Human Protein Atlas. NSUN6 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. NSUN6 subcellular location (ICC-IF): Localized to the Golgi apparatus..
- Human Protein Atlas. NSUN6 antibody validation summary (1 antibodies).
- RNA m5C methylation orchestrates BLCA progression via macrophage reprogramming. Journal of cellular and molecular medicine 2023 — PMC10424284.
- Downregulated m⁵C regulator NSUN6 enhances proliferation, migration and affects immune regulation in ovarian cancer. Scientific reports 2026 — PMC13454582.
- NSUN6 overexpression promotes malignant phenotypes and predicts poor prognosis in clear cell renal cell carcinoma. Cancer cell international 2026 — PMC13270792.
- NSUN6-mediated 5-methylcytosine modification of NDRG1 mRNA promotes radioresistance in cervical cancer. Molecular cancer 2024 — PMC11225205.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15164054 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NSUN6 (tRNA (cytosine(72)-C(5))-methyltransferase NSUN6). Expected localisation Granular cytoplasm in most tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A13404-3). Validated IHC antibody: A13404-3 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Bronchus (Ciliated cells (cell body)); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet A13404-3); 2. Fixation: Image fixative and duration unreported (datasheet A13404-3); 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 A13404-3); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A13404-3); 8. Primary antibody: Rabbit anti-NSUN6, 2-5 μg/ml (datasheet A13404-3); 9. Primary incubation: Overnight at 4 °C (datasheet A13404-3). Watch for: Fixation: Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific); Caveat: Staining has medium consistency with RNA data (HPA tissue IHC); Isoform / epitope: No isoforms annotated; one 1–469 chain (UniProt); Regulation: No expression regulator annotated (UniProt). Evidence: UniProt Q8TEA1, HPA tissue IHC · Approved, HPA ICC-IF · Golgi apparatus., Datasheet A13404-3, PMC13454582, PMC13270792. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NSUN6/immunohistochemistry-ihc-guide.