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NDUFAF1 / Complex I intermediate-associated protein 30, mitochondrial · IHC design guide
Design Immunohistochemistry for NDUFAF1
Plan paraffin IHC for NDUFAF1 using catalog antibody A10145 at 2–5 μg/ml (datasheet A10145). Interpret granular cytoplasmic staining (HPA tissue IHC) alongside its mitochondrial matrix association (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NDUFAF1 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 (HPA tissue IHC); mitochondrial matrix face of the inner membrane (UniProt) | |
| Staining pattern | Granular cytoplasmic staining across many cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A10145) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | None in HPA (detected in all 44 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep tissue fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A10145) | |
| Caveat | Ubiquitous staining limits tissue-negative controls (HPA tissue IHC) | |
| Regulation | Ubiquitous expression (UniProt) | |
| Isoform / epitope | No isoforms annotated; mature chain spans aa 25–327 (UniProt) |
Section 1
Recommended NDUFAF1 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: A10145) is accompanied by one published pancreatic tissue microarray protocol (PMC4414165).
| Sample | Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A10145) |
| Fixation | Image fixative and duration unreported (datasheet A10145); 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 A10145); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A10145) |
| Primary antibody | Rabbit anti-NDUFAF1, 2-5 μg/ml (datasheet A10145) |
| Primary incubation | Overnight at 4 °C (datasheet A10145) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A10145) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | NDUFAF1-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression with a granular pattern. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet: A10145); the published protocol boiled slides in EDTA at pH 8.0 for 15 minutes (PMC4414165).
Section 2
What Is the Expected NDUFAF1 Staining Pattern?
NDUFAF1 is mitochondrial, peripherally associated with the matrix face of the inner membrane, and has no transmembrane segment (UniProt Q9Y375). In paraffin-section IHC, expect granular cytoplasmic staining across many cell types (HPA tissue IHC: ubiquitous granular pattern; Supported reliability). HPA reports high staining in several glandular, respiratory epithelial, hematopoietic, and neuronal populations (HPA tissue IHC).
| Granular cytoplasmic staining in glandular cells of adrenal gland, appendix, or breast. | Consistent with the reported pattern and high staining in these cells (HPA tissue IHC). Chromogenic granules alone cannot prove mitochondrial identity (standard IHC interpretation). |
| Predominantly nuclear or sharply plasma-membrane staining. | Discordant with mitochondrial matrix-side localization (UniProt Q9Y375) and HPA's granular cytoplasmic pattern (HPA tissue IHC); assess nonspecific staining before scoring it as NDUFAF1. |
| Staining confined to an unexpected cell population while an expected high-staining population is blank. | Consider cross-reactivity or endogenous detection activity (standard IHC practice). HPA reports ubiquitous expression and supplies no negative cell population, so cell identity alone cannot establish an artifact (HPA tissue IHC). |
| Uniform cytoplasmic haze obscures granules and tissue boundaries. | This cannot be confidently read as the reported granular pattern (HPA tissue IHC). Diffuse background can arise from nonspecific binding or detection chemistry (standard IHC practice). |
| No discernible staining in an expected high-staining population. | Treat the run as unresolved before calling NDUFAF1 absent: HPA reports high staining in named populations (HPA tissue IHC), while failed detection is also possible (standard IHC practice). |
💡Expected NDUFAF1 appearanceA convincing positive is discernible granular cytoplasmic staining in an HPA high-staining cell population (HPA tissue IHC); dominant nuclear or crisp surface staining is discordant with mitochondrial localization (UniProt Q9Y375).
| Compartment and optical resolution | NDUFAF1 lies on the matrix face of the mitochondrial inner membrane (UniProt Q9Y375). Read chromogenic granularity as compatible with that location, not as proof of an individual organelle (standard IHC interpretation). |
| Tissue and cell context | HPA records high staining in adrenal, appendix, and breast glandular cells; bronchial respiratory epithelium; marrow hematopoietic cells; and selected neurons (HPA tissue IHC). It lists no negative tissue here (HPA tissue IHC). |
| Strength of pattern evidence | The tissue pattern is Supported, with medium agreement between antibody staining and RNA expression (HPA tissue IHC). Both HPA039933 and HPA040064 have Supported IHC status (HPA antibodies); neither is listed as Enhanced. |
| Processing and epitope information | The annotated mature chain spans residues 25–327; no transmembrane segment, annotated isoforms, or glycosylation sites are supplied (UniProt Q9Y375). Antibody epitope and target-specific retrieval or fixation sensitivity are unreported in this record. |
| IF/ICC Q&A | Does IF/ICC support the compartment call? HPA lists mitochondria as supported and cytosol as uncertain, with images from A-431, HEK293, and U2OS (HPA subcellular). That evidence supports a mitochondrial interpretation; it does not define an IHC-P protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected high-staining tissue is blank. | The detection run may have failed; a blank slide alone does not overturn HPA's high-staining report (HPA tissue IHC; standard IHC practice). | Check tissue preservation and control-slide staining, then review the IHC-validated antibody's dilution, retrieval, and detection steps (standard IHC practice). |
| Signal appears chiefly in nuclei or at cell borders. | That distribution conflicts with matrix-side mitochondrial localization (UniProt Q9Y375); nonspecific binding or detection artifact is possible (standard IHC practice). | Compare a matched negative control, examine counterstain and tissue morphology, and withhold a positive call until cytoplasmic granularity is resolved (standard IHC practice). |
| Broad brown haze makes granules hard to distinguish. | The haze may reflect excess reagent, inadequate washing, or nonspecific detection (standard IHC practice), rather than HPA's granular cytoplasmic pattern (HPA tissue IHC). | Review antibody dilution, washing, blocking, and chromogen development with matched controls (standard IHC practice). |
| Only one unexpected cell group stains strongly. | Cell-restricted background or cross-reactivity is possible (standard IHC practice); ubiquitous expression means an unexpected cell type is not automatically negative (UniProt Q9Y375). | Check morphology and negative controls, then compare the same cell population with HPA's cell-resolved tissue observations before assigning specificity (HPA tissue IHC; standard IHC practice). |
| Brown signal persists when primary antibody is omitted. | Endogenous detection activity or nonspecific secondary-system staining may contribute (standard IHC practice). | Use the appropriate enzyme-blocking and detection-system controls; interpret the primary-antibody slide only after control background is understood (standard IHC practice). |
| A granular signal is present, but mitochondrial attribution remains uncertain. | IHC morphology has limited organelle resolution (standard IHC interpretation). HPA's ICC-IF record supports mitochondria but labels cytosolic localization uncertain (HPA subcellular). | Score the IHC finding as compatible with mitochondrial localization and keep the cytosolic claim qualified; consult the separate IF/ICC guide for an orthogonal localization experiment (HPA subcellular). |
Experiment evidence
IHC & IF images showing NDUFAF1 staining
A10145 · IHCIHC analysis of NDUFAF1 using anti-NDUFAF1 antibody (A10145). NDUFAF1 was detected in a paraffin-embedded section of human breast cancer tis
A10145 · IHCIHC analysis of NDUFAF1 using anti-NDUFAF1 antibody (A10145). NDUFAF1 was detected in a paraffin-embedded section of human liver cancer tiss
A10145 · IFIF analysis of NDUFAF1 using anti-NDUFAF1 antibody (A10145). NDUFAF1 was detected in an immunocytochemical section of T47D cells. Enzyme ant
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000137806-NDUFAF1/tissue/cerebral+cortex#imid_11574359
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000137806-NDUFAF1/tissue/colon#imid_11574277
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000137806-NDUFAF1/tissue/liver#imid_11574278
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000137806-NDUFAF1/tissue/kidney#imid_11574271
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA039933; green = NDUFAF1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA039933; green = NDUFAF1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NDUFAF1 IHC & IF
🧪Run breast tissue first and assess staining in glandular cells (HPA: High in breast glandular cells; A10145 tissue-IHC caption: human breast cancer section). HPA detects NDUFAF1 in all 44 scored tissues, so use no-primary and isotype controls for the negative; adjacent nonglandular cells can be compared for lower staining, but should not be presumed antigen-negative (HPA: no negative tissues; UniProt Q9Y375: ubiquitous).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: NDUFAF1 is detected in all 44 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 NDUFAF1 in A-431, HEK293, U2OS, with annotated localisation: Mitochondria (supported), Cytosol (uncertain) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only section, a matched rabbit IgG control of the primary antibody’s clonality, and NDUFAF1 knockout material as a biological negative if available (A10145 tissue-IHC caption: rabbit primary and biotinylated goat anti-rabbit secondary). For the breast section, block endogenous peroxidase and check endogenous biotin background in the biotin-based DAB workflow (A10145 tissue-IHC caption: streptavidin–biotin detection with DAB).
⚠️Feasibility: The supplied evidence reports no target-specific fixation window or fixation effect, and the selected A10145 tissue-IHC caption does not state the fixative. That caption uses EDTA retrieval at pH 8.0 on a paraffin section, but one example does not establish that retrieval is required (A10145 tissue-IHC caption). The evidence does not establish whether frozen sections or IF/ICC are easier; in breast IHC, assess background from the caption’s biotin-based detection before scoring (A10145 tissue-IHC caption).
HPA tissue IHC evidence for NDUFAF1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — 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: NDUFAF1 is detected in all 44 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced NDUFAF1 IHC Tips
Troubleshoot NDUFAF1 staining in paraffin sections using the catalog antibody’s IHC result and the reported mitochondrial localisation (caption A10145; UniProt Q9Y375).
Which retrieval condition should I try first for weak NDUFAF1 staining?
Start with heat-mediated retrieval in EDTA at pH 8.0 (datasheet A10145). This condition accompanied NDUFAF1 detection in a paraffin section of human breast cancer, followed by 2 μg/ml primary antibody overnight at 4°C (caption A10145). If staining remains weak, compare retrieval times on adjacent sections while keeping antibody concentration, detection chemistry and development time constant; record tissue damage alongside signal (standard IHC practice). Evaluate any gain in granular cytoplasmic staining against a section processed without primary antibody, since the expected pattern is mitochondrial and HPA reports granular cytoplasmic tissue staining (UniProt Q9Y375; HPA tissue IHC).
How should I troubleshoot possible fixation effects in paraffin sections?
Target-specific fixation sensitivity for NDUFAF1 is unknown: the selected paraffin-section caption does not state a fixative (caption A10145). Record the fixation method and duration for each specimen, then compare sections prepared under known conditions using the same EDTA pH 8.0 retrieval and 2 μg/ml primary antibody conditions (datasheet A10145; caption A10145; standard IHC practice). Include a comparably processed positive-control section and a no-primary control to distinguish loss of detectable signal from detection background (standard IHC practice). Do not attribute a difference to NDUFAF1-specific fixation sensitivity without a controlled comparison, because the supplied IHC evidence establishes no such effect (caption A10145).
Should I score nuclear or diffuse cytoplasmic staining as NDUFAF1?
Prioritise granular cytoplasmic staining when reviewing the chromogenic section: HPA describes a ubiquitous granular cytoplasmic IHC pattern, and UniProt places NDUFAF1 in mitochondria, peripherally on the matrix face of the inner membrane (HPA tissue IHC; UniProt Q9Y375). Record diffuse cytoplasmic staining separately, since HPA lists cytosol localisation as uncertain while mitochondrial localisation is supported (HPA subcellular). Treat predominantly nuclear staining as discordant with the supplied localisation evidence and compare it with no-primary background (UniProt Q9Y375; HPA subcellular; standard IHC practice). Inspect intact cells at comparable section thickness and development time before assigning a compartment or comparing specimens (standard IHC practice).
Can processing or isoforms explain inconsistent NDUFAF1 staining?
The supplied record lists 0 isoforms and a processed chain spanning residues 25–327; these facts alone do not identify the catalog antibody’s epitope (UniProt Q9Y375). Ask which sequence the antibody recognises before attributing different IHC patterns to precursor processing, and avoid assuming that the unreported epitope survives retrieval equally across specimens (UniProt Q9Y375; standard IHC practice). NDUFAF1 has no annotated transmembrane segment or glycosylation sites, while phosphoserine 318 is reported; none establishes an epitope-dependent staining effect for this antibody (UniProt Q9Y375). Compare adjacent sections under matched retrieval and detection conditions and document any reproducible compartment change (standard IHC practice).
How can IF help check the chromogenic NDUFAF1 pattern?
Use IF as a separate localisation check: HPA reports supported mitochondrial localisation, uncertain cytosol localisation, and ICC/IF images in A-431, HEK293 and U2OS cells (HPA subcellular). Multiplex NDUFAF1 with a validated marker for the cell type being assessed and, where useful, a mitochondrial marker; establish each channel’s specificity with single-stain controls (standard IF practice; UniProt Q9Y375 localisation). Choose spectrally separated fluorophores and inspect unstained tissue autofluorescence before interpreting puncta (standard IF practice). Because NDUFAF1 lies on the matrix face of the inner membrane, optimise permeabilisation to give antibody access to that compartment; the supplied evidence specifies no IF fixation or permeabilisation settings (UniProt Q9Y375; HPA subcellular).
What should I change when DAB background obscures granular staining?
Check a section processed without primary antibody, then inspect whether the DAB signal follows tissue edges, damaged regions or the expected granular cytoplasmic distribution (standard IHC practice; HPA tissue IHC). The reported workflow used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, a biotinylated secondary for 30 minutes at 37°C, and streptavidin–biotin detection with DAB (caption A10145). Treat a peroxidase block and DAB development control as general chromogenic IHC steps, rather than NDUFAF1-specific validation (standard IHC practice). If controls implicate reagent background, optimise blocking, washing or primary concentration one variable at a time while retaining a positive-control section (standard IHC practice).
How should I compare NDUFAF1 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored compartment as granular cytoplasm and identify the cell population before measuring signal, consistent with the reported tissue pattern and mitochondrial localisation (HPA tissue IHC; UniProt Q9Y375). For chromogenic sections, record the percentage of positive cells and intensity categories, or calculate an H-score from 0–300 using the same thresholds across specimens (standard IHC practice). Normalise positive-cell counts to the number of eligible intact cells, or positive-cell density to analysed tissue area in mm²; exclude folds and necrotic areas by a prespecified rule (standard IHC practice). Keep section preparation, imaging and DAB development comparable, and report cell composition because NDUFAF1 expression is broadly distributed (standard IHC practice; HPA tissue IHC).
Which findings support a true NDUFAF1-positive IHC result?
A credible result shows reproducible granular cytoplasmic signal in intact cells, consistent with HPA tissue IHC and NDUFAF1’s mitochondrial location (HPA tissue IHC; UniProt Q9Y375). HPA reports high staining in breast glandular cells and several other listed cell populations, but its tissue IHC reliability is “Supported” with medium consistency between staining and RNA expression (HPA tissue IHC). Flag predominantly nuclear signal, staining confined to section edges or necrosis, and signal in the no-primary control for investigation before calling a specimen positive (UniProt Q9Y375; standard IHC practice). Check endogenous peroxidase contribution with an appropriate detection control, then compare the suspect section with a consistently processed positive control (standard IHC practice).
Boster reagents
Best NDUFAF1 / Complex I intermediate-associated protein 30, mitochondrial IHC Antibodies
A10145 has IHC images from human paraffin-embedded tissue and an IF image from T47D cells (A10145 image captions); its catalog lists human, mouse and rat reactivity (catalog reactivity).
Real IHC data
Anti-NDUFAF1 Antibody ®
Cat # A10145
A10145 will render with an IHC figure from a human paraffin-embedded breast cancer section; its captions also document human liver cancer, lung cancer and lymphoma sections and IF in T47D cells (A10145 image captions). M10145-1 will render as a human-reactive antibody listed for IHC and IF/ICC, with no IHC or IF image in the payload (M10145-1 catalog applications/reactivity and image alts).
Which to pick: Choose A10145 for paraffin-section IHC: its figure documents EDTA retrieval at pH 8.0, 2 μg/ml primary antibody and DAB detection; the fixative is unreported (A10145 IHC image caption). Choose A10145 for IF/ICC when an image-backed option is needed; M10145-1 is a rabbit monoclonal listed for human IHC and IF/ICC, but has no image evidence in this payload (A10145 IF image caption; M10145-1 catalog host/clone, applications and image alts). For mouse or rat samples, A10145 is the catalog-listed option; the supplied tissue IHC captions show human sections only (A10145 catalog reactivity and IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9Y375 (CIA30_HUMAN, Complex I intermediate-associated protein 30, mitochondrial).
- Human Protein Atlas. NDUFAF1 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. NDUFAF1 subcellular location (ICC-IF): Localized to the cytosol and mitochondria..
- Human Protein Atlas. NDUFAF1 antibody validation summary (2 antibodies).
- Identification of NDUFAF1 in mediating K-Ras induced mitochondrial dysfunction by a proteomic screening approach. Oncotarget 2015 — PMC4414165.
- PubMed PMID:10810093 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:11935339 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NDUFAF1 (Complex I intermediate-associated protein 30, mitochondrial). Expected localisation Granular cytoplasm (HPA tissue IHC); mitochondrial matrix face of the inner membrane (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A10145). Validated IHC antibody: A10145 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative None in HPA (detected in all 44 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A10145); 2. Fixation: Image fixative and duration unreported (datasheet A10145); 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 A10145); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A10145); 8. Primary antibody: Rabbit anti-NDUFAF1, 2-5 μg/ml (datasheet A10145); 9. Primary incubation: Overnight at 4 °C (datasheet A10145). Watch for: Fixation: Keep tissue fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A10145); Caveat: Ubiquitous staining limits tissue-negative controls (HPA tissue IHC); Isoform / epitope: No isoforms annotated; mature chain spans aa 25–327 (UniProt); Regulation: Ubiquitous expression (UniProt). Evidence: UniProt Q9Y375, HPA tissue IHC · Supported, HPA ICC-IF · cytosol and mitochondria., Datasheet A10145, PMC4414165. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NDUFAF1/immunohistochemistry-ihc-guide.