This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
ACAD9 / Complex I assembly factor ACAD9, mitochondrial · IHC design guide
Design Immunohistochemistry for ACAD9
Plan chromogenic ACAD9 IHC in paraffin sections with the catalog antibody at 2–5 μg/ml (datasheet A05223-3). Assess granular cytoplasmic staining by cell type, using high-staining glandular cells and low-staining skeletal myocytes as references (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ACAD9 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 inner membrane association (UniProt) | |
| Staining pattern | Granular cytoplasmic staining; high in glandular cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A05223-3) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Adipose tissue+2 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); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A05223-3) | |
| Caveat | Skeletal myocytes may stain low despite higher tissue expression (HPA tissue IHC; UniProt) | |
| Regulation | Higher expression in heart, muscle and CNS (UniProt) | |
| Isoform / epitope | No isoforms annotated; mature chain begins at residue 38 (UniProt) |
Section 1
Recommended ACAD9 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is provided separately (datasheet: A05223-3). The published protocols below cover femur (PMC13039524) and human cerebellum and muscle (PMC3073726).
| Sample | Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A05223-3) |
| Fixation | Image fixative and duration unreported (datasheet A05223-3); 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 A05223-3); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A05223-3) |
| Primary antibody | Rabbit anti-ACAD9, 2-5 μg/ml (datasheet A05223-3) |
| Primary incubation | Overnight at 4 °C (datasheet A05223-3) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A05223-3) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ACAD9-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression with granular pattern. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet: A05223-3). Use citrate retrieval when reproducing the femur protocol (PMC13039524).
Section 2
What Is the Expected ACAD9 Staining Pattern?
ACAD9 is associated with the mitochondrial inner membrane and has no annotated transmembrane segment (UniProt Q9H845 topology). In paraffin sections, expect granular cytoplasmic staining in cells such as colon glandular cells and cerebellar granular-layer cells (HPA tissue IHC: High). HPA describes the overall IHC pattern as granular cytoplasmic, but rates its tissue evidence Supported, with medium agreement with RNA data and external verification pending (HPA tissue IHC).
| Granular cytoplasmic staining in colon or appendix glandular cells, with nuclei largely unstained. | This fits the reported IHC distribution: those glandular cells stain at a High level, and the overall pattern is granular cytoplasmic (HPA tissue IHC). Granules are consistent with mitochondrial localization (UniProt Q9H845; HPA subcellular), but chromogenic morphology alone does not confirm each granule is a mitochondrion. |
| Predominantly nuclear staining, or a crisp cell-surface outline, replaces cytoplasmic granules. | Treat this as a compartment mismatch and investigate artefact or nonspecific binding; the reported pattern is cytoplasmic and granular (HPA tissue IHC), while ACAD9 is mitochondrial and has no annotated transmembrane segment (UniProt Q9H845 topology). Assess the pattern against a control section before scoring it as ACAD9. |
| Strong staining appears in adipocytes, ovarian stromal cells, or smooth muscle cells. | Those specific cell populations are reported as Not detected (HPA tissue IHC). Check morphology and controls: unexpected staining could reflect cross-reactivity or endogenous detection activity (general IHC practice). The HPA observation applies to the named cells, not every cell in their respective tissues. |
| A uniform brown haze covers cells and surrounding tissue, obscuring granules. | The result cannot be confidently scored against the expected granular pattern (HPA tissue IHC). Diffuse haze can arise from nonspecific detection or residual endogenous activity (general chromogenic IHC practice); review the control without primary antibody and the blocking and wash steps. |
| No signal appears in colon glandular cells or cerebellar granular-layer cells. | Both are reported High by tissue IHC (HPA tissue IHC), so absence of signal warrants a run-level check. Verify tissue identity, antibody and detection steps, and an appropriate positive control (general IHC practice). HPA's Supported rating and pending external verification limit certainty about any single specimen (HPA tissue IHC). |
💡Expected ACAD9 appearanceCall a section positive when identifiable HPA-reported cells show clear, predominantly granular cytoplasmic staining—potentially strong in colon glandular cells—while predominantly nuclear, cell-surface, or uniform diffuse staining prompts review as a possible false positive (HPA tissue IHC; UniProt Q9H845 localization; general IHC interpretation).
| Which compartment should guide scoring? | Use granular cytoplasmic staining as the brightfield criterion (HPA tissue IHC). Mitochondrial localization supports that interpretation (UniProt Q9H845; HPA subcellular); ordinary chromogenic resolution does not establish precise inner-membrane placement within each stained cell. |
| How much should tissue intensity vary? | HPA reports High staining in several epithelial and neural cell populations, but Low staining in skeletal-muscle myocytes (HPA tissue IHC). UniProt reports higher overall expression in skeletal muscle (UniProt Q9H845 tissue specificity); avoid using either statement alone as a universal IHC intensity threshold. |
| How strong is the IHC validation? | The tissue profile is Supported, with medium staining–RNA consistency and external verification pending (HPA tissue IHC). HPA037716 is listed as IHC Supported; the supplied antibody record gives no IHC status for HPA046720 (HPA antibodies). Interpret unexpected patterns with controls rather than treating validation as definitive. |
| Do processing or modifications set an IHC retrieval rule? | UniProt annotates a mature chain beginning at residue 38 and several modified residues (UniProt Q9H845 processing and PTMs). Neither record supplies an antibody epitope or target-specific retrieval response; choose and assess retrieval through the catalog antibody's IHC-P instructions and controls (general IHC practice). |
| Does IF/ICC support the same location? | Yes: HPA reports an enhanced mitochondrial ICC-IF location, with images from A-431 and U2OS, and lists ICC as Enhanced for both supplied antibodies (HPA subcellular; HPA antibodies). This supports a mitochondrial interpretation; IF/ICC methods and optimization belong to its separate guide. |
| Situation | Likely cause | Next action |
|---|---|---|
| No granular staining in a reported High cell population. | The run may have weak detection, or the sampled section may not contain the intended cells (general IHC practice); HPA reports High staining only for specified populations (HPA tissue IHC). | Identify the cells on the counterstain, check the positive-control section, then review retrieval, catalog-antibody dilution, and detection against the IHC-P instructions (general IHC practice). Do not infer an ACAD9-specific fixation effect from these sources. |
| Brown signal persists without primary antibody. | Endogenous enzyme activity or nonspecific detection can produce chromogenic signal (general IHC practice). Such signal does not establish ACAD9 expression. | Compare the no-primary control with the test section; check the detection reagent, endogenous-activity block appropriate to that detection chemistry, and washes (general IHC practice). Score ACAD9 only where interpretable granular cellular signal remains. |
| Diffuse haze masks cytoplasmic granules. | Nonspecific binding or excess detection background may obscure the HPA-reported granular pattern (HPA tissue IHC; general IHC practice). | Review blocking and wash steps and assess the catalog-antibody dilution within its IHC-P guidance (general IHC practice). Compare background with a control section before changing the interpretation. |
| Nuclear or cell-surface signal dominates. | That compartment conflicts with reported granular cytoplasmic IHC staining and mitochondrial localization (HPA tissue IHC; UniProt Q9H845). Artefact or off-target binding is possible. | Check cellular landmarks and counterstain, compare positive and no-primary controls, and reassess antibody and detection conditions (general IHC practice). Do not score the mismatched pattern as confirmed ACAD9. |
| Adipocytes or smooth muscle cells appear strongly positive. | HPA reports those cell types as Not detected (HPA tissue IHC); cross-reactivity or endogenous activity is possible (general IHC practice). | Confirm cell identity, inspect the no-primary control, and compare with a reported High cell population processed in the same run (general IHC practice; HPA tissue IHC). Treat persistent unexpected staining as unresolved. |
| Skeletal-muscle myocytes stain weakly despite an expectation of high expression. | HPA records Low IHC staining in myocytes, whereas UniProt describes higher overall expression in skeletal muscle (HPA tissue IHC; UniProt Q9H845 tissue specificity). These observations measure different things. | Use the observed myocyte pattern as a qualified comparison, and assess a reported High IHC cell population and run controls before calling the assay unsuccessful (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing ACAD9 staining
A05223-3 · IHCIHC analysis of ACAD9 using anti-ACAD9 antibody (A05223-3). ACAD9 was detected in a paraffin-embedded section of human breast cancer tissue.
A05223-3 · IHCIHC analysis of ACAD9 using anti-ACAD9 antibody (A05223-3). ACAD9 was detected in a paraffin-embedded section of human larynx squamous cell
A05223-3 · IFIF analysis of ACAD9 using anti-ACAD9 antibody (A05223-3). ACAD9 was detected in a paraffin-embedded section of human breast cancer tissue.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000177646-ACAD9/tissue/cerebral+cortex#imid_10070580
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000177646-ACAD9/tissue/colon#imid_10070514
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000177646-ACAD9/tissue/liver#imid_10070515
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000177646-ACAD9/tissue/kidney#imid_10070516
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA037716; green = ACAD9, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA037716; green = ACAD9, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ACAD9 IHC & IF
🧪Run cerebellum first and score staining in cells of the granular layer, where ACAD9 is reported at High level (HPA: cerebellum, cells in granular layer). Run adipose tissue as a negative comparator, scoring adipocytes reported as Not detected (HPA: adipose tissue, adipocytes); on the cerebellum slide, unstained areas can indicate background, but no other cell type is established as ACAD9-negative by the supplied evidence.
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ACAD9 in A-431, U2OS, with annotated localisation: Mitochondria (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; a rabbit IgG isotype control matched to the primary antibody’s clonality and concentration where known; and ACAD9-knockout material or a validated peptide-block control as a biological specificity check (tissue-IHC caption: rabbit primary; standard IHC control practice). Check endogenous peroxidase background in cerebellum before interpreting DAB staining (standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A05223-3 tissue-IHC caption does not state a fixative (A05223-3 tissue-IHC caption). The caption uses heat-mediated EDTA retrieval at pH 8.0, but does not establish that retrieval is required; it provides a paraffin-section IHC starting condition (A05223-3 tissue-IHC caption). The supplied evidence does not establish whether frozen sections or IF are easier; dense cells in the cerebellar granular layer may make individual-cell scoring difficult (HPA: cerebellum, cells in granular layer; standard histology practice).
HPA tissue IHC evidence for ACAD9
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — Medium consistency between antibody staining and RNA expression data. Pending external verification.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
Section 3
Advanced ACAD9 IHC Tips
Troubleshoot ACAD9 staining in paraffin sections by checking retrieval, cell level localisation, controls, and scoring before interpreting signal intensity.
What retrieval should I start with for ACAD9 in paraffin sections?
Start with heat mediated retrieval in EDTA at pH 8.0 (datasheet A05223-3). The selected tissue IHC image used this retrieval before incubation with 2 μg/ml antibody overnight at 4°C (caption A05223-3). If staining is weak, compare a small retrieval time series while holding section thickness, antibody concentration, and detection constant (standard IHC practice). Include a section processed without primary antibody to identify signal introduced by the detection system (standard IHC practice). Judge retrieval by whether granular cytoplasmic staining becomes clearer in expected cells without a parallel increase in diffuse background (HPA: granular cytoplasmic profile; standard IHC practice).
Could fixation explain weak or uneven ACAD9 staining?
ACAD9 specific sensitivity to fixation is unknown because the selected tissue IHC caption does not state a fixative (caption A05223-3). Record the fixative, fixation duration, tissue thickness, and processing history for each specimen before comparing staining (standard IHC practice). On a new specimen series, hold retrieval at EDTA pH 8.0 and antibody concentration at 2 μg/ml while comparing documented processing conditions (caption A05223-3; standard IHC practice). Examine internal cells on each section to distinguish a processing gradient from a biological difference (standard IHC practice). Do not assign weak signal to an ACAD9 specific fixation effect without a controlled comparison (standard IHC practice).
Where should convincing ACAD9 chromogenic staining appear?
Look for a granular cytoplasmic pattern rather than a continuous nuclear or cell surface signal (HPA: general cytoplasmic expression with granular pattern). ACAD9 localises to mitochondria and is essentially associated with the inner membrane despite having no annotated transmembrane segment (UniProt Q9H845 localisation and topology). Inspect the pattern at sufficient magnification to distinguish intracellular granules from pigment, debris, and precipitated chromogen (standard IHC practice). High staining in cerebellar granular layer cells and colon glandular cells provides tissue level context, subject to the atlas assessment of supported reliability and pending external verification (HPA: tissue IHC). A mitochondrial pattern supports localisation but does not establish antibody specificity by itself (standard IHC practice).
How should I assess epitope accessibility without a mapped antibody epitope?
Treat the epitope as unmapped: the supplied record lists 0 isoforms but gives no antibody binding sequence (UniProt Q9H845; caption A05223-3). The annotated mature chain spans residues 38–621, so an epitope in the removed N terminal region would require separate validation rather than an assumed staining pattern (UniProt Q9H845 processing; standard IHC practice). Annotated modified residues include lysine 41, lysine 92, threonine 478, and residue 521 (UniProt Q9H845 modified residues). Their effect on this antibody is unknown because its epitope is unspecified (caption A05223-3). Compare retrieval conditions and, where available, an independently validated antibody against a disclosed region before attributing staining differences to processing or modification (standard IHC practice).
How can I investigate ACAD9 localisation by IF alongside tissue IHC?
Use IF as a separate assay requiring its own antibody validation; the selected evidence documents chromogenic staining in a paraffin section (caption A05223-3). Multiplex ACAD9 with a marker identifying the expected cell type, and use a mitochondrial marker to assess its reported location (HPA: tissue IHC and subcellular localisation; standard IF practice). Choose spectrally separated fluorophores after checking tissue autofluorescence in an unstained control, especially before assigning faint puncta to ACAD9 (standard IF practice). ACAD9 is associated with the mitochondrial inner membrane without an annotated transmembrane segment, but the antibody epitope's accessible side is unspecified (UniProt Q9H845; caption A05223-3). Optimise permeabilisation empirically for epitope access while checking mitochondrial morphology and single channel controls (standard IF practice).
What should I change when ACAD9 DAB staining looks diffuse?
First compare the stained section with a no primary control and inspect whether signal follows tissue edges or damaged regions (standard IHC practice). The selected image used 10% goat serum block, 2 μg/ml primary antibody overnight at 4°C, and a peroxidase conjugated secondary for 30 minutes at 37°C (caption A05223-3). A peroxidase block and controlled DAB development are general chromogenic IHC steps, not ACAD9 specific validation (standard IHC practice). If diffuse staining persists, titrate the primary antibody and shorten chromogen development one variable at a time (standard IHC practice). Retain granular cytoplasmic staining as the pattern to evaluate while monitoring whether background falls (HPA: tissue IHC profile; standard IHC practice).
How should I quantify ACAD9 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and scoring region before examining treatment groups (standard IHC practice). Record the percentage of positive cells and intensity categories, or calculate an H score from those categories; for spatial questions, report positive cell density per mm² of viable tissue (standard IHC practice). Keep retrieval, antibody concentration, DAB development, imaging, and threshold settings consistent across specimens (standard IHC practice). Normalise scores to the number of evaluable cells or viable tissue area, and report excluded necrotic or folded regions (standard IHC practice). Score the granular cytoplasmic signal within each cell population rather than treating every brown deposit as ACAD9 (HPA: granular cytoplasmic profile; standard IHC practice).
How do I distinguish credible ACAD9 staining from artefact?
A credible result combines intracellular granular cytoplasmic staining with appropriate cell context and a clean no primary control (HPA: tissue IHC profile; standard IHC practice). ACAD9 is mitochondrial and essentially inner membrane associated, so dominant nuclear or cell surface staining warrants investigation (UniProt Q9H845 localisation and topology). Compare suspect deposits with section edges, necrotic areas, and sites of endogenous peroxidase activity before calling them positive (standard IHC practice). The atlas reports high staining in appendix glandular cells but no detected staining in adipocytes; its tissue IHC reliability is supported with external verification pending (HPA: tissue IHC). Use those observations as contextual controls, then confirm unexpected patterns with an independent specificity test (standard IHC practice).
Boster reagents
Best ACAD9 / Complex I assembly factor ACAD9, mitochondrial IHC Antibodies
A05223-3 has IHC images from four human paraffin tissue samples and an IF image from human breast cancer tissue (catalog image captions).
Real IHC data
Anti-ACAD9 Antibody ®
Cat # A05223-3
A05223-3 is listed for human IHC and IF (catalog applications and reactivity). Its IHC captions show breast cancer, larynx squamous cell carcinoma, liver cancer, and lung adenocarcinoma tissue; its IF caption shows breast cancer tissue (catalog image captions).
Which to pick: For tissue IHC, choose A05223-3: its human breast cancer image documents paraffin sections, EDTA retrieval at pH 8.0, and 2 μg/ml primary antibody (A05223-3 IHC image caption). For IF, A05223-3 has a human breast cancer paraffin-section image at 5 μg/ml; ICC validation is unreported (A05223-3 IF image caption; catalog applications). No cross-species option is documented: A05223-3 lists human reactivity only, its clonality is unreported, and its paraffin-section captions do not report the fixative (catalog reactivity and clone field; A05223-3 image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9H845 (ACAD9_HUMAN, Complex I assembly factor ACAD9, mitochondrial).
- Human Protein Atlas. ACAD9 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. ACAD9 subcellular location (ICC-IF): Localized to the mitochondria..
- Human Protein Atlas. ACAD9 antibody validation summary (2 antibodies).
- Moonlighting cytosolic function of ACAD9: suppression of TRAF6-mediated osteoclastogenesis and protection against osteoporosis. Cell death & disease 2026 — PMC13039524.
- Identification and characterization of new long chain acyl-CoA dehydrogenases. Molecular genetics and metabolism 2011 — PMC3073726.
- PubMed PMID:12359260 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ACAD9 (Complex I assembly factor ACAD9, mitochondrial). Expected localisation Granular cytoplasm (HPA tissue IHC); mitochondrial inner membrane association (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A05223-3). Validated IHC antibody: A05223-3 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A05223-3); 2. Fixation: Image fixative and duration unreported (datasheet A05223-3); 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 A05223-3); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A05223-3); 8. Primary antibody: Rabbit anti-ACAD9, 2-5 μg/ml (datasheet A05223-3); 9. Primary incubation: Overnight at 4 °C (datasheet A05223-3). Watch for: Fixation: Keep fixation consistent across paraffin sections (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 A05223-3); Caveat: Skeletal myocytes may stain low despite higher tissue expression (HPA tissue IHC; UniProt); Isoform / epitope: No isoforms annotated; mature chain begins at residue 38 (UniProt); Regulation: Higher expression in heart, muscle and CNS (UniProt). Evidence: UniProt Q9H845, HPA tissue IHC · Supported, HPA ICC-IF · mitochondria., Datasheet A05223-3, PMC13039524, PMC3073726. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ACAD9/immunohistochemistry-ihc-guide.