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NDUFS3 / NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, mitochondrial · IHC design guide
Design Immunohistochemistry for NDUFS3
Plan chromogenic NDUFS3 IHC in paraffin sections using the IHC-validated antibody at 2–5 μg/ml (datasheet A05867-2). Compare cytoplasmic staining in high-staining kidney tubules with undetected prostate glandular cells (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NDUFS3 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 (HPA tissue IHC); mitochondrial inner membrane (UniProt) | |
| Staining pattern | General cytoplasmic staining across tissues (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A05867-2) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Oral mucosa+2 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 | Prostate glandular cells may show no staining (HPA tissue IHC) | |
| Regulation | Expression varies by tissue and cell type (HPA tissue IHC) | |
| Isoform / epitope | 2 isoforms; their epitope coverage is unspecified (UniProt) |
Section 1
Recommended NDUFS3 IHC & IF Protocols
The catalog antibody protocol (datasheet A05867-2) is paired with published NDUFS3 IHC methods for melanoma tissue microarrays (PMC12669717) and formalin-fixed tissue sections (PMC6385215).
| Sample | Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A05867-2) |
| Fixation | Image fixative and duration unreported (datasheet A05867-2); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A05867-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A05867-2) |
| Primary antibody | Rabbit anti-NDUFS3, 2-5 μg/ml (datasheet A05867-2) |
| Primary incubation | Overnight at 4 °C (datasheet A05867-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A05867-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | NDUFS3-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA at pH 8.0 (datasheet A05867-2); the published TE-T retrieval at pH 8.0 is another documented condition (PMC6385215).
Section 2
What Is the Expected NDUFS3 Staining Pattern?
NDUFS3 is a core Complex I subunit of the mitochondrial inner membrane and has no transmembrane segment (UniProt O75489: function and topology). In paraffin-section IHC, expect cytoplasmic staining in documented positive cells, including cardiomyocytes, kidney tubule cells, and pancreatic exocrine glandular cells (HPA: High in each). HPA describes general cytoplasmic expression and rates its tissue IHC reliability Enhanced, with medium consistency between staining and RNA data (HPA: tissue IHC).
| Granular or finely distributed cytoplasmic chromogen in cardiomyocytes or kidney tubule cells (HPA: High in both). | This fits the expected mitochondrial-associated pattern (UniProt O75489: inner membrane; HPA: mainly mitochondrial in ICC-IF). Routine chromogenic IHC may show cytoplasmic signal without resolving individual mitochondria (general IHC practice). |
| Predominantly nuclear or cell-surface staining, with little cytoplasmic signal. | Treat this as discordant with the principal location (UniProt O75489: inner membrane; HPA: mitochondria supported). HPA also lists nuclear bodies as an uncertain additional ICC-IF location; that observation alone does not establish nuclear IHC as the expected pattern (HPA: subcellular). |
| Strong staining in prostate glandular cells while an adjacent documented positive control stains appropriately (HPA: prostate glandular cells Not detected). | Investigate nonspecific antibody binding or endogenous chromogen activity (general IHC practice). HPA's Not detected call applies to the listed cells and assay observations; it is not proof that every prostate specimen must be negative (HPA: tissue IHC). |
| Uniform color across stroma, empty spaces, and cells, obscuring cell boundaries. | Diffuse background limits interpretation of a cell-specific result (general IHC practice). Check the detection-only control and background-reduction steps before scoring intensity; HPA's cytoplasmic profile cannot validate a uniformly stained section (HPA: tissue IHC). |
| No signal in cardiomyocytes, kidney tubule cells, or pancreatic exocrine glandular cells (HPA: High in each). | A negative run is difficult to interpret until a known-positive section works. Review section integrity, retrieval, antibody application, and detection with matched controls (general IHC practice); HPA's Enhanced rating does not guarantee signal under every local assay condition (HPA: tissue IHC). |
💡Expected NDUFS3 appearanceCall a positive result when documented positive cells show discernible cytoplasmic, mitochondrial-associated chromogen, potentially granular at adequate resolution (HPA: High in cardiomyocytes and kidney tubule cells; UniProt O75489: inner membrane); predominant nuclear staining or uniform background warrants investigation (HPA: mitochondria supported; general IHC practice).
| Compartment and optical resolution (UniProt O75489: inner membrane; HPA: general cytoplasmic IHC profile). | Score the chromogenic pattern at the cytoplasmic level; a paraffin section need not resolve an inner-membrane outline to agree with mitochondrial localization (general IHC practice). |
| Choice of comparison cells (HPA: High in heart cardiomyocytes and kidney tubules; Not detected in oral squamous cells). | Use a documented high-staining cell population to judge assay performance. Interpret a Not detected population as an HPA observation, not a universal biological absence (HPA: tissue IHC). |
| Antibody evidence (HPA: HPA004484 and CAB034894 IHC Enhanced; HPA: tissue IHC reliability Enhanced). | Enhanced supports the reported IHC pattern, while HPA notes only medium consistency with RNA expression. Check the actual antibody's validation before transferring a pattern claim to it (HPA: tissue IHC and antibodies). |
| Protein processing and variants (UniProt O75489: mature chain 37–264; two isoforms). | The record identifies processing and two isoforms, but supplies no antibody epitope or isoform-specific staining result. Do not assign an unexpected IHC pattern to either factor without antibody-specific evidence (UniProt O75489: processing and isoforms). |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive cells are blank (HPA: High in cardiomyocytes or kidney tubule cells). | A failed staining run or inadequate assay sensitivity is possible (general IHC practice). | Confirm tissue preservation and detection controls; review the antibody's IHC-P instructions, retrieval, and working dilution as general assay checks. No NDUFS3-specific fixation sensitivity is established here (HPA: tissue IHC; general IHC practice). |
| Chromogen is predominantly nuclear (HPA: mitochondria supported in ICC-IF). | The pattern conflicts with the principal NDUFS3 compartment; HPA calls nuclear bodies an uncertain additional ICC-IF location (HPA: subcellular). | Compare a documented positive cell population and a detection-only control, then assess antibody specificity before calling nuclear IHC positive (HPA: tissue IHC; general IHC practice). |
| A nominally negative cell population stains strongly (HPA: prostate glandular cells Not detected). | Nonspecific binding or endogenous detection activity may contribute (general IHC practice). HPA's negative call is limited to its observed samples and cells (HPA: tissue IHC). | Check a detection-only control and the stained cell identity; compare with a documented positive cell population before interpreting the new signal (general IHC practice; HPA: tissue IHC). |
| Diffuse background obscures cytoplasmic detail. | Excess reagent, incomplete blocking, or endogenous detection activity can obscure localization (general IHC practice). | Assess detection-only and background controls, then optimize blocking, antibody concentration, and washes within the IHC-P workflow (general IHC practice). Score only when cellular signal can be distinguished from background. |
| Different tissues show unequal intensity (HPA: High in pancreas exocrine cells; Low in smooth muscle cells). | HPA reports different staining levels by cell population, so equal intensity is not an expected acceptance criterion (HPA: tissue IHC). | Score identified cell populations separately and compare each with its documented HPA category. Use a high-staining population to assess run performance before interpreting a low-staining one (HPA: tissue IHC; general IHC practice). |
| Q: Should an IF/ICC image match the chromogenic IHC appearance? | HPA supports mitochondria as the main ICC-IF location, while paraffin IHC is described as generally cytoplasmic (HPA: subcellular and tissue IHC). | A: Compare compartment-level localization, allowing for each method's resolution and detection readout (general IHC/IF practice). Treat HPA's nuclear-body and principal-piece ICC-IF locations as uncertain, not required IHC features (HPA: subcellular). |
Experiment evidence
IHC & IF images showing NDUFS3 staining
A05867-2 · IHCIHC analysis of NDUFS3 using anti-NDUFS3 antibody (A05867-2). NDUFS3 was detected in a paraffin-embedded section of human breast cancer tiss
A05867-2 · IHCIHC analysis of NDUFS3 using anti-NDUFS3 antibody (A05867-2). NDUFS3 was detected in a paraffin-embedded section of human ovarian cancer tis
M05867-2 · IHCHuman kidney was stained with anti-NDUFS3 rabbit antibody
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000213619-NDUFS3/tissue/cerebral+cortex#imid_1605092
HPA · StomachHuman Protein Atlas IHC, Stomach. Source: https://www.proteinatlas.org/ENSG00000213619-NDUFS3/tissue/stomach+2#imid_1604976
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000213619-NDUFS3/tissue/colon#imid_1605010
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000213619-NDUFS3/tissue/liver#imid_1605027
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA004484; green = NDUFS3, blue = nucleus, red = microtubules.
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA004484; green = NDUFS3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NDUFS3 IHC & IF
🧪Run appendix first: glandular cells should stain strongly (HPA: High in appendix glandular cells). Use oral mucosa squamous epithelial cells as the negative tissue (HPA: Not detected); on the appendix slide, assess adjacent nonglandular cells for background rather than assuming they are target negative (HPA: glandular-cell result only).
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Oral mucosa (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show NDUFS3 in MCF-7, Sperm, with annotated localisation: Mitochondria (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and concentration-matched rabbit IgG isotype controls, plus NDUFS3 knockout material or a validated immunizing-peptide competition control where available (selected-SKU caption: rabbit primary antibody). For chromogenic detection, block endogenous peroxidase and inspect the appendix control for residual background (selected-SKU caption: HRP/DAB detection).
⚠️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 caption: fixative not stated). The demonstrated tissue-IHC conditions used heat-mediated EDTA retrieval at pH 8.0; retrieval dependence has not been established (selected-SKU caption: EDTA retrieval). IF may help assess the expected mitochondrial localization (HPA subcellular: mitochondria supported; UniProt O75489: inner mitochondrial membrane), but the supplied evidence does not establish that IF or frozen sections are easier than paraffin IHC, or identify an appendix-specific artefact.
HPA tissue IHC evidence for NDUFS3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — 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 |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuropil | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | High | Protein (IHC) | HPA → |
Section 3
Advanced NDUFS3 IHC Tips
Troubleshoot NDUFS3 chromogenic IHC in paraffin sections by checking retrieval, compartment-specific staining, controls and scoring before interpreting biological differences.
What retrieval should I try first when NDUFS3 staining is weak?
Start with heat-mediated retrieval in EDTA pH 8.0 for paraffin-section IHC (datasheet A05867-2). The selected antibody's tissue image used that retrieval before incubation at 2 μg/ml overnight at 4°C (datasheet A05867-2). If signal remains weak, compare a longer or shorter heating interval on adjacent sections while holding antibody concentration and chromogen development constant (standard IHC practice). Include a documented high-staining cell population, such as kidney tubule cells, so retrieval changes have a reference (HPA: High in kidney tubules). Record tissue morphology alongside staining intensity, because excessive heating can damage sections and complicate interpretation (standard IHC practice).
Could fixation explain variable NDUFS3 staining across paraffin sections?
The selected tissue-IHC caption identifies a paraffin-embedded section but does not state its fixative (datasheet A05867-2). NDUFS3-specific sensitivity to fixation is therefore unknown from the supplied tissue-IHC evidence (datasheet A05867-2). Compare sections with documented fixation histories using the same EDTA pH 8.0 retrieval and 2 μg/ml primary concentration (datasheet A05867-2; standard IHC practice). Keep section thickness, detection and DAB development comparable, and assess morphology before attributing a signal difference to fixation (standard IHC practice). A fixation effect needs a controlled comparison; staining differences between unrelated tissue regions alone cannot establish one (standard IHC practice).
Where should convincing NDUFS3 staining appear in chromogenic IHC?
Expect staining in the cytoplasmic region that contains mitochondria, assessed at the resolution available in chromogenic sections (UniProt O75489: inner-mitochondrial-membrane localisation; HPA: general cytoplasmic tissue expression). NDUFS3 has no annotated transmembrane segment, so a crisp cell-surface outline is not its expected pattern (UniProt O75489: topology). Compare the candidate signal with high-staining cardiomyocytes or kidney tubule cells in suitable control sections (HPA: High in heart muscle cardiomyocytes and kidney tubules). Diffuse nuclear staining without a corresponding cytoplasmic component warrants a detection and morphology check (UniProt O75489: localisation; standard IHC practice). HPA's additional nuclear-body localisation is uncertain and should not override its supported mitochondrial localisation (HPA subcellular: supported mitochondria; uncertain nuclear bodies).
Can this antibody distinguish NDUFS3 isoforms or reveal a masked epitope?
The record lists 2 NDUFS3 isoforms, but the supplied antibody caption does not identify its epitope or establish isoform discrimination (UniProt O75489: isoforms; datasheet A05867-2). The annotated mature chain spans residues 37–264, which alone cannot show whether this antibody recognizes a shared or isoform-specific region (UniProt O75489: processing). Avoid assigning differing tissue stains to particular isoforms without mapped epitope and isoform-specific validation (standard IHC practice). If staining is absent, first review retrieval, primary incubation and control-section performance under the documented conditions (datasheet A05867-2; standard IHC practice). The record annotates 0 glycosylation sites and 0 modified residues, but does not establish epitope accessibility after processing (UniProt O75489: annotations).
How should I follow up an IHC result with multiplex immunofluorescence?
Use IF as a separate validation experiment: the selected antibody evidence documents chromogenic paraffin-section IHC, not an IF staining condition (datasheet A05867-2). Pair NDUFS3 with a validated marker of the cell population being assessed, such as cardiomyocytes in heart muscle, and check whether both signals occur in the intended cells (HPA: High in cardiomyocytes; standard IF practice). Choose fluorophores after measuring the section's autofluorescence and reserve a spectrally distinct channel for each marker (standard IF practice). Because NDUFS3 is mitochondrial and lacks an annotated transmembrane segment, optimise permeabilisation for antibody access to the intracellular epitope; its exact side is unreported (UniProt O75489: localisation and topology; standard IF practice). Include single-label controls to assess bleed-through (standard IF practice).
How can I reduce diffuse or misleading DAB background?
Check whether the unwanted colour persists in a no-primary control before changing the NDUFS3 antibody concentration (standard IHC practice). The documented workflow used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and a peroxidase-linked secondary for 30 minutes at 37°C (datasheet A05867-2). Quench endogenous peroxidase and compare shorter DAB development on adjacent sections if colour appears without primary antibody (standard IHC practice). Keep washing and section handling consistent while changing one condition at a time (standard IHC practice). Judge whether the remaining signal follows a cytoplasmic mitochondrial distribution in appropriate cells (UniProt O75489: localisation; HPA: general cytoplasmic expression).
What is a defensible way to quantify NDUFS3 IHC? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region of interest before scoring, then record both staining intensity and the percentage of positive cells (standard IHC practice). An H-score combines intensity categories with their positive-cell fractions on a 0–300 scale; apply identical thresholds across compared sections (standard IHC practice). For heterogeneous sections, report scores by cell type rather than averaging glandular, stromal and other compartments together (standard IHC practice). Normalise counts or positive area to the corresponding number of evaluable cells or tissue area, and exclude folds and necrotic regions consistently (standard IHC practice). Interpret differences against matched controls because HPA reports only medium consistency between antibody staining and RNA data (HPA: Enhanced reliability description).
When is an apparent NDUFS3-positive region likely to be artefact?
A credible signal should occupy the cytoplasmic region of plausible cells, consistent with mitochondrial localisation and the tissue-IHC expression profile (UniProt O75489: inner-membrane localisation; HPA: general cytoplasmic expression). High staining in kidney tubules or cardiomyocytes offers a useful comparison, while absent staining in oral-mucosa squamous cells is an expression reference rather than an absolute exclusion rule (HPA: tissue IHC). Treat staining confined to nuclei, section edges or necrotic areas as suspect until morphology and controls support it (UniProt O75489: localisation; standard IHC practice). Check a no-primary section for endogenous enzyme or detection-related colour before scoring DAB deposits as NDUFS3 (standard IHC practice). Require the same compartment and cell-type pattern across intact replicate sections (standard IHC practice).
Boster reagents
Best NDUFS3 / NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, mitochondrial IHC Antibodies
Two anti-NDUFS3 antibodies have IHC images from human and rat tissue (catalog image captions); both list human, mouse, and rat reactivity, and one lists IF (catalog applications/reactivity).
Real IHC data
Anti-NDUFS3 Antibody ®
Cat # A05867-2
Real IHC data
Anti-NDUFS3 Rabbit Monoclonal Antibody
Cat # M05867-2
A05867-2 has IHC images of paraffin-embedded human breast cancer, human ovarian cancer, and rat testis sections (A05867-2 image captions). M05867-2 has an IHC image of human kidney and lists IF among its applications; no IF image is supplied (M05867-2 image caption; catalog applications/images).
Which to pick: For paraffin-section IHC, choose A05867-2: its captions document 2 μg/ml antibody after heat retrieval in EDTA at pH 8.0; the fixative is unreported (A05867-2 image captions). For IF, choose M05867-2 (catalog applications); for ICC, M05867 lists that application but has no IHC or IF image in the payload (catalog applications/images). Both rendered SKUs list human, mouse, and rat reactivity, but the supplied IHC images show human and rat tissue for A05867-2 and human tissue for M05867-2 (catalog reactivity; image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O75489 (NDUS3_HUMAN, NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, mitochondrial).
- Human Protein Atlas. NDUFS3 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. NDUFS3 subcellular location (ICC-IF): Mainly localized to the mitochondria. In addition localized to the nuclear bodies and principal piece..
- Human Protein Atlas. NDUFS3 antibody validation summary (2 antibodies).
- Alpha-ketoglutarate alleviates acute pancreatitis by restoring energy metabolism. iScience 2026 — PMC13059122.
- NDUFS3 promotes proliferation via glucose metabolism reprogramming inducing AMPK phosphorylating PRPS1 to increase the purine nucleotide synthesis in melanoma. Cell death and differentiation 2025 — PMC12669717.
- Development of a Curcumin-Loaded Nanomicelles-Injectable Sustained-Release Hydrogel System for Modulating Oxidative Stress to Alleviate Tendinopathy. International journal of molecular sciences 2026 — PMC12941043.
- Inducing cancer indolence by targeting mitochondrial Complex I is potentiated by blocking macrophage-mediated adaptive responses. Nature communications 2019 — PMC6385215.
- PubMed PMID:9647766 — UniProt-cited evidence.
- PubMed PMID:10967146 — UniProt-cited evidence.
- PubMed PMID:10931946 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NDUFS3 (NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, mitochondrial). Expected localisation Cytoplasmic staining (HPA tissue IHC); mitochondrial inner membrane (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A05867-2). Validated IHC antibody: A05867-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Oral mucosa. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A05867-2); 2. Fixation: Image fixative and duration unreported (datasheet A05867-2); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: EDTA pH 8.0 (datasheet A05867-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A05867-2); 8. Primary antibody: Rabbit anti-NDUFS3, 2-5 μg/ml (datasheet A05867-2); 9. Primary incubation: Overnight at 4 °C (datasheet A05867-2). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Prostate glandular cells may show no staining (HPA tissue IHC); Isoform / epitope: 2 isoforms; their epitope coverage is unspecified (UniProt); Regulation: Expression varies by tissue and cell type (HPA tissue IHC). Evidence: UniProt O75489, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the mitochondria. In addition localized to the…, Datasheet A05867-2, PMC12669717, PMC6385215. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NDUFS3/immunohistochemistry-ihc-guide.