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NDUFB9 / NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 9 · IHC design guide
Design Immunohistochemistry for NDUFB9
Plan NDUFB9 paraffin IHC around the granular cytoplasmic staining seen in most tissues (HPA tissue IHC). Cardiomyocytes and kidney tubule cells show high staining (HPA tissue IHC); the A08623-1 IHC-P starting concentration is 5 μg/mL (datasheet).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NDUFB9 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 | Granular cytoplasm in cardiomyocytes and kidney tubule cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Adipose tissue+4 more · see all | |
| Negative control | Bone marrow+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 | Antibody staining has low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | No stimulus-specific regulation annotated (UniProt) | |
| Isoform / epitope | No annotated isoforms; chain spans residues 2–179 (UniProt) |
Section 1
Recommended NDUFB9 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with a published NDUFB9 staining workflow for FFPE slides (PMC13376752).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A08623-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-NDUFB9, 5 μg/mL (datasheet A08623-1) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | NDUFB9-positive staining in adipocytes of adipose tissue (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression with a granular expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page antigen retrieval).
Section 2
What Is the Expected NDUFB9 Staining Pattern?
NDUFB9 is a mitochondrial inner-membrane Complex I accessory subunit with no annotated transmembrane segment (UniProt Q9Y6M9). In paraffin-section IHC, expect granular cytoplasmic staining in many tissues, including cardiomyocytes, kidney tubule cells, adipocytes and several glandular cell populations scored High by HPA (HPA: tissue IHC). HPA rates its tissue staining Approved but reports low consistency with RNA expression, so interpret intensity alongside controls and tissue morphology (HPA: reliability).
| Granular cytoplasmic staining in cardiomyocytes or kidney tubule cells, with preserved tissue detail. | This fits HPA's granular cytoplasmic profile and High staining scores for those cells (HPA: tissue IHC). It is compatible with NDUFB9's mitochondrial location (UniProt Q9Y6M9; HPA: subcellular IF). Chromogenic granules alone do not resolve individual mitochondria (general IHC practice). |
| Predominantly nuclear staining or a sharp cell-surface outline replaces cytoplasmic granules. | That compartment differs from the reported mitochondrial location and granular cytoplasmic IHC profile (UniProt Q9Y6M9; HPA: tissue IHC). Check controls, morphology and detection conditions before treating it as NDUFB9 localisation (general IHC practice). |
| Strong staining appears in bone-marrow hematopoietic cells or lymph-node germinal-center cells. | HPA reports NDUFB9 as Not detected in those specific cell populations (HPA: tissue IHC). Review cell identification and controls; cross-reactivity or endogenous detection activity is possible, but an HPA Not detected score does not prove every specimen must be negative (general IHC practice). |
| Color spreads diffusely across tissue, including spaces without recognizable cells. | That distribution does not match HPA's granular cytoplasmic profile (HPA: tissue IHC). Background from the detection workflow is a possibility; use the appropriate negative control and assess whether the color tracks tissue structure (general IHC practice). |
| No staining is visible in an otherwise intact heart-muscle or kidney section. | HPA scores cardiomyocytes and kidney tubule cells High, making them useful reference populations (HPA: tissue IHC). Recheck the assay and a concurrently stained positive control before interpreting the absence as biological; HPA staining is a reference observation, not a guarantee for every specimen (general IHC practice). |
💡Expected NDUFB9 appearanceA positive IHC result is granular cytoplasmic staining in expected cells such as cardiomyocytes or kidney tubule cells, which HPA scores High; dominant nuclear staining or color outside cells calls for investigation (HPA: tissue IHC; UniProt Q9Y6M9 localisation; general IHC practice).
| Where should chromogenic staining appear? | NDUFB9 is assigned to the mitochondrial inner membrane (UniProt Q9Y6M9), while tissue IHC shows a granular cytoplasmic pattern (HPA: tissue IHC). Interpret the chromogenic pattern at the resolution of the section; do not claim that individual granules prove inner-membrane localisation (general IHC practice). |
| How should tissue-to-tissue intensity be used? | HPA scores cardiomyocytes, kidney tubule cells and adipocytes High, breast glandular cells Medium, and liver hepatocytes Low (HPA: tissue IHC). These are observed cell-specific scores, useful for comparison within the documented context; they are not a universal intensity threshold for all specimens (general IHC practice). |
| How strong is the tissue-staining evidence? | HPA marks tissue IHC Approved while noting low consistency between antibody staining and RNA expression (HPA: reliability). The listed antibody HPA042768 is IHC Approved; no Enhanced IHC status is supplied (HPA: antibody validation). Keep unexpected staining provisional until controls and morphology support it (general IHC practice). |
| Do processing or sequence features predict the IHC pattern? | UniProt lists a chain spanning residues 2–179, no annotated transmembrane segment, and no annotated isoforms (UniProt Q9Y6M9). These facts do not identify the antibody epitope or establish antigen-retrieval requirements or fixation sensitivity; use them only as sequence context. |
| Does IF/ICC support the localisation? | Yes. HPA reports supported mitochondrial localisation by ICC-IF and lists A-431, U-251MG, U2OS and KOLF2.1J among cell lines with images (HPA: subcellular IF). This supports a mitochondrial interpretation; the IF/ICC staining procedure belongs in its separate guide. |
| Situation | Likely cause | Next action |
|---|---|---|
| A known-positive heart or kidney section has no visible signal. | The section conflicts with HPA's High scores for cardiomyocytes or kidney tubule cells; an assay failure is possible, but the image alone cannot identify its cause (HPA: tissue IHC; general IHC practice). | Check a concurrently stained positive control, antibody application, antigen-retrieval record, detection reagents and counterstain visibility; adjust workflow steps using the catalog antibody's IHC-P instructions (general IHC practice). |
| Color is predominantly nuclear or outlines cell membranes. | The compartment conflicts with mitochondrial localisation and HPA's granular cytoplasmic IHC profile (UniProt Q9Y6M9; HPA: tissue IHC). Nonspecific staining or misread morphology is possible (general IHC practice). | Reassess the cells at higher magnification, compare a negative control and review antibody and detection conditions before scoring NDUFB9 positivity (general IHC practice). |
| A population HPA scores Not detected stains strongly. | For example, HPA scores bone-marrow hematopoietic cells Not detected; cell misidentification, cross-reactivity or endogenous detection activity could explain a discrepancy (HPA: tissue IHC; general IHC practice). | Confirm the cell population morphologically, inspect a negative control and compare an HPA High reference population processed in the same run (HPA: tissue IHC; general IHC practice). |
| A diffuse haze obscures otherwise granular staining. | The haze differs from HPA's granular cytoplasmic profile; nonspecific binding or detection background may contribute (HPA: tissue IHC; general IHC practice). | Compare tissue-free areas and a negative control, then review blocking, washes and chromogen development within the validated IHC workflow (general IHC practice). |
| A low-scored tissue appears weaker than a high-scored tissue. | HPA scores liver hepatocytes Low and cardiomyocytes High, so a difference in these cells can reflect the reported tissue pattern (HPA: tissue IHC). Technical variation can also affect an IHC comparison (general IHC practice). | Compare the documented cell populations and run controls before assigning biological meaning to intensity; do not apply one tissue's HPA score to every cell in that tissue (HPA: tissue IHC; general IHC practice). |
| IHC granules appear less discrete than mitochondrial IF signal. | HPA reports granular cytoplasmic tissue IHC and supported mitochondrial ICC-IF localisation; the methods present localisation at different visual scales (HPA: tissue IHC; HPA: subcellular IF; general IHC practice). | Score the IHC section for the expected cytoplasmic pattern and cell type. Use the separate IF/ICC guide for fluorescence-specific interpretation (HPA: tissue IHC; HPA: subcellular IF). |
Experiment evidence
IHC & IF images showing NDUFB9 staining
A08623-1 · IHCImmunohistochemistry of LYRM3 in rat liver tissue with LYRM3 antibody at 5 μg/mL.
A08623-1 · IFImmunofluorescence of LYRM3 in rat liver tissue with LYRM3 antibody at 20 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000147684-NDUFB9/tissue/cerebral+cortex#imid_11196415
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000147684-NDUFB9/tissue/colon#imid_11196205
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000147684-NDUFB9/tissue/liver#imid_11196206
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000147684-NDUFB9/tissue/kidney#imid_11196215
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA042768; green = NDUFB9, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA042768; green = NDUFB9, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NDUFB9 IHC & IF
🧪Run kidney first: tubular cells should stain strongly (HPA: High in kidney cells in tubules). Use bone marrow hematopoietic cells as a negative comparator (HPA: Not detected in bone marrow hematopoietic cells). On the kidney slide, cells confirmed as internal negatives should lack specific granular cytoplasmic staining; the supplied HPA rows do not establish a negative kidney cell type (HPA: kidney row identifies tubular cells only; UniProt Q9Y6M9: mitochondrial inner membrane).
Positive control tissue: Adipose tissue (Adipocytes, HPA High)
Negative control tissue: Bone marrow (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show NDUFB9 in A-431, U-251MG, U2OS, KOLF2.1J, with annotated localisation: Mitochondria (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; an isotype control matched to the primary antibody’s host and immunoglobulin class, or host IgG for a polyclonal primary; and knockout tissue or peptide-blocked primary as a biological specificity control (standard IHC practice). Quench endogenous peroxidase in kidney sections and check for endogenous biotin background if using biotin-based detection (standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A08623-1 rat-liver IHC caption does not state its fixative (selected-SKU caption). Antigen-retrieval dependence is also unreported; optimize retrieval on matched paraffin sections while checking kidney tubular staining and background (HPA: High in kidney cells in tubules; standard IHC practice). For IF/ICC, mitochondrial localization has supporting images, but the supplied evidence does not establish that frozen sections or IF are easier than paraffin IHC; assess tissue autofluorescence and use a mitochondrial reference stain if testing IF (HPA: mitochondria supported; standard IF practice).
HPA tissue IHC evidence for NDUFB9
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low 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 |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | High | Protein (IHC) | HPA → |
| Kidney | Cells in tubules | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | Basal cells | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NDUFB9 IHC Tips
Troubleshoot NDUFB9 staining in paraffin sections by checking retrieval, compartment, tissue controls and scoring consistency (UniProt Q9Y6M9; HPA tissue IHC).
What retrieval conditions should I start with for NDUFB9 in paraffin sections?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval setting). Let sections cool in buffer, then compare staining with a retrieval-omitted section and a tissue control processed in the same run (standard IHC practice). Seek granular cytoplasmic staining rather than diffuse nuclear signal, since NDUFB9 localizes to the mitochondrial inner membrane and tissue staining is described as granular cytoplasmic (UniProt Q9Y6M9; HPA tissue IHC). If staining remains weak, adjust heating duration in a small comparison while keeping detection and exposure to chromogen consistent; document any changed conditions before comparing samples (standard IHC practice).
Could fixation explain weak or uneven NDUFB9 staining?
Target-specific fixation sensitivity is unknown: the selected rat liver IHC caption reports 5 µg/mL antibody but does not state a fixative (selected A08623-1 tissue-IHC caption). Record fixative, fixation duration and processing history for each paraffin specimen before comparing signal intensity (standard IHC practice). Compare similarly processed sections in one staining run, including a positive tissue control, because uneven handling can confound chromogenic staining (standard IHC practice). If staining varies, test retrieval and antibody concentration on serial sections from the same block before attributing a difference to NDUFB9 abundance (standard IHC practice). Do not infer a fixation effect from mitochondrial localization or reported tissue patterns (UniProt Q9Y6M9; HPA tissue IHC).
Where should convincing NDUFB9 staining appear in a paraffin section?
Expect granular cytoplasmic staining consistent with mitochondria, rather than a nuclear or cell-surface pattern (HPA tissue IHC; HPA subcellular; UniProt Q9Y6M9). NDUFB9 is assigned to the mitochondrial inner membrane and has no annotated transmembrane segment, so chromogenic resolution alone cannot establish its position within that membrane (UniProt Q9Y6M9). Compare the pattern within intact cells against a same-run positive control, and check whether granules follow cell boundaries rather than tissue folds or pigment (standard IHC practice). HPA reports high staining in kidney tubular cells and heart cardiomyocytes, while liver hepatocytes show low staining; choose comparison tissue accordingly and avoid treating weak liver signal alone as assay failure (HPA tissue IHC).
Could epitope placement or processing change how this antibody stains NDUFB9?
Check the antibody’s documented immunogen or epitope before interpreting a missing signal; its epitope is not supplied here (catalog evidence supplied). UniProt lists 0 isoforms and a mature chain spanning residues 2–179, with no annotated transmembrane segment (UniProt Q9Y6M9). It also records N-acetylalanine at residue 2 and phosphoserine at residue 85; these annotations do not establish whether this antibody recognizes either site (UniProt Q9Y6M9; catalog evidence supplied). If two antibodies with documented distinct epitopes become available, compare their granular cytoplasmic patterns on adjacent sections while holding retrieval and detection constant (standard IHC practice; HPA tissue IHC).
How should I adapt NDUFB9 localization checks to multiplex IF?
Treat IF as a separate assay and establish fixation and permeabilisation conditions with the IF antibody’s own validation data; none are supplied for this catalog antibody (catalog evidence supplied). NDUFB9 localizes to mitochondria, but its antibody epitope and which side of the inner membrane it faces are unspecified, so compare gentle and stronger permeabilisation conditions empirically (HPA subcellular; UniProt Q9Y6M9; catalog evidence supplied; standard IF practice). Multiplex with a marker identifying the expected cell type, such as a validated tubular-cell marker when assessing kidney, and inspect channels individually before interpreting overlap (HPA tissue IHC; standard IF practice). Select a fluorophore channel with low tissue autofluorescence and include single-channel controls for bleed-through (standard IF practice).
How can I separate NDUFB9 signal from chromogenic background?
Inspect a no-primary control and a same-run positive tissue control before interpreting diffuse brown deposition as NDUFB9 (standard IHC practice). Include a peroxidase block when using an HRP–DAB workflow, and compare the no-primary control for endogenous enzyme activity (standard IHC practice). Check folds, section edges, damaged areas and pigment under the microscope; these can mimic cellular staining or exaggerate local DAB intensity (standard IHC practice). Genuine staining should favor a granular cytoplasmic pattern, consistent with the HPA tissue description and mitochondrial localization (HPA tissue IHC; HPA subcellular). If background rises with antibody concentration, titrate around the documented 5 µg/mL rat liver condition without assuming it transfers to every tissue (selected A08623-1 tissue-IHC caption; standard IHC practice).
What is a defensible way to score NDUFB9 in IHC sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and an analysis region before scoring, then measure the percentage of cells with granular cytoplasmic staining or apply a consistent intensity-based H-score (HPA tissue IHC; standard IHC practice). For spatial comparisons, report positive-cell density per mm² alongside the number of evaluable cells or tissue area, using identical thresholds across sections (standard IHC practice). Normalize comparisons to the relevant cell population or viable tissue area, rather than raw brown area across specimens with different cellularity (standard IHC practice). Record retrieval, antibody concentration, chromogen development and imaging settings, and score a same-run control to detect staining drift (standard IHC practice). Interpret intensity cautiously because HPA labels tissue IHC reliability Approved while reporting low consistency with RNA expression (HPA tissue IHC).
Which patterns argue for true NDUFB9 staining rather than an artefact?
Give more weight to granular cytoplasmic staining in intact cells, consistent with mitochondrial localization, than to isolated nuclear, luminal or extracellular deposits (HPA tissue IHC; HPA subcellular; UniProt Q9Y6M9). Check cell identity: HPA reports high staining in kidney tubular cells and cardiomyocytes, but no detection in bone marrow hematopoietic cells or smooth muscle cells (HPA tissue IHC). Discount signals confined to section edges, folds or necrotic areas, and compare them with a no-primary control for endogenous peroxidase staining (standard IHC practice). A convincing result should reproduce its cellular pattern across comparable sections and controls; staining intensity alone is insufficient because HPA reports low consistency between antibody staining and RNA expression (standard IHC practice; HPA tissue IHC).
Boster reagents
Best NDUFB9 / NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 9 IHC Antibodies
A08623-1 has rat liver IHC and IF images (catalog image captions); both antibodies list human, mouse and rat reactivity (catalog reactivity).
Real IHC data
Anti-LYRM3 NDUFB9 Antibody
Cat # A08623-1
A08623-1 shows rat liver IHC at 5 μg/mL and IF at 20 μg/mL (catalog image captions). M08623-3 lists IHC and ICC/IF applications, but has no IHC or IF image in the payload (catalog applications and image captions).
Which to pick: Choose A08623-1 for paraffin-section IHC: it lists IHC-P and shows rat liver staining, although the fixative is unreported (catalog applications; A08623-1 IHC image caption). For IF/ICC, A08623-1 has a rat liver IF image, while M08623-3 lists ICC/IF and is monoclonal, with no image supplied (catalog applications, clone and image captions). Both list human, mouse and rat reactivity; the supplied tissue images document rat liver only (catalog reactivity; A08623-1 image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9Y6M9 (NDUB9_HUMAN, NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 9).
- Human Protein Atlas. NDUFB9 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. NDUFB9 subcellular location (ICC-IF): Localized to the mitochondria..
- Human Protein Atlas. NDUFB9 antibody validation summary (1 antibodies).
- Complex I protein NDUFB9 is a metabolic vulnerability in triple negative breast cancer brain metastases. Nature communications 2026 — PMC13376752.
- PubMed PMID:10077726 — UniProt-cited evidence.
- PubMed PMID:10944468 — UniProt-cited evidence.
- PubMed PMID:11042152 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NDUFB9 (NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 9). Expected localisation Granular cytoplasm in most tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A08623-1 at 5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Adipose tissue (Adipocytes); negative Bone marrow. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet A08623-1); 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-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-NDUFB9, 5 μg/mL (datasheet A08623-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Antibody staining has low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: No annotated isoforms; chain spans residues 2–179 (UniProt); Regulation: No stimulus-specific regulation annotated (UniProt). Evidence: UniProt Q9Y6M9, HPA tissue IHC · Approved, HPA ICC-IF · mitochondria., Datasheet A08623-1, PMC13376752. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NDUFB9/immunohistochemistry-ihc-guide.