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ACOT9 / Acyl-coenzyme A thioesterase 9, mitochondrial · IHC design guide
Design Immunohistochemistry for ACOT9
Plan ACOT9 paraffin-section IHC around granular cytoplasmic staining with a mitochondrial pattern (HPA tissue IHC). Use lung macrophages as a high-staining reference (HPA tissue IHC), and titrate the IHC-validated antibody within 1:50–1:200 (datasheet).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ACOT9 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 with a mitochondrial pattern (HPA tissue IHC) | |
| Staining pattern | Granular cytoplasmic staining in lung macrophages (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Adrenal gland+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 sections (standard IHC practice; not target-specific) | |
| Caveat | Adipocytes: not detected; lung macrophages: high (HPA tissue IHC) | |
| Regulation | Staining regulation is not established (UniProt) | |
| Isoform / epitope | 4 isoforms; mature chain begins at residue 22; epitope effects unknown (UniProt) |
Section 1
Recommended ACOT9 IHC & IF Protocols
The catalog antibody protocol is followed by two published ACOT9 IHC protocols for paraffin sections (PMC13374139; PMC13335382).
| Sample | Paraffin-embedded human colon carcinoma tissue; fixative not specified (datasheet A14147-1) |
| Fixation | Image fixative and duration unreported (datasheet A14147-1); 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-ACOT9, 1:50-1:200 (datasheet A14147-1) |
| 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 | ACOT9-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression with granular, mitochondrial pattern in all tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 heat retrieval at 95–98 °C for 20 min (page antigen-retrieval setting); adjust if tissue staining requires optimization.
Section 2
What Is the Expected ACOT9 Staining Pattern?
ACOT9 is annotated in the mitochondrial matrix and inner membrane, with no transmembrane segment (UniProt Q9Y305: location and topology). In tissue IHC, expect granular cytoplasmic staining, including in adrenal glandular cells, bronchial respiratory epithelial cells and lung macrophages (HPA: tissue IHC profile; High in these cells). HPA rates the tissue pattern Supported, with medium consistency between staining and RNA expression (HPA: reliability).
| Granular cytoplasmic staining in adrenal glandular cells, bronchial respiratory epithelial cells or lung macrophages (HPA: High in each). | This fits the reported mitochondrial pattern (HPA: tissue IHC profile; UniProt Q9Y305: mitochondrial location). Compare cell types within the section; HPA reports low tissue specificity, so staining beyond these examples can still be plausible (HPA: RNA specificity). |
| Predominantly nuclear or sharply cell-surface staining in tissue IHC. | Treat this as a compartment mismatch requiring review, rather than calling it ACOT9-positive: HPA describes granular cytoplasm in tissue IHC (HPA: tissue IHC profile). HPA's separate ICC-IF observations include plasma membrane and nucleoplasm, so those images do not settle an IHC discrepancy (HPA: subcellular ICC-IF). |
| Strong staining in adipocytes, skeletal myocytes or vaginal squamous epithelial cells. | These cells were not detected in the supplied tissue IHC record (HPA: adipose tissue, skeletal muscle and vagina). Check cell identification and controls; cross-reactivity or endogenous detection activity are possible explanations for an unexpected signal (general IHC practice), not established properties of ACOT9. |
| Broad, even colour across cells, stroma and blank areas. | A diffuse field does not match the granular cytoplasmic tissue profile (HPA: tissue IHC profile). Review background and detection controls before scoring cells; excess reagent signal or endogenous detection activity can produce nonspecific colour (general IHC practice). |
| No staining in a section containing a reported high-staining cell population. | First confirm that the expected cells are present: for example, lung macrophages are High, whereas this does not describe every lung cell (HPA: lung tissue IHC). Then check the IHC detection run and its controls (general IHC practice); absence alone cannot establish loss of ACOT9. |
💡Expected ACOT9 appearanceCall positive when the relevant cells show clear granular cytoplasmic staining, especially a population reported High by HPA; diffuse background, isolated nuclear colour or staining confined to reported undetected cells is suspect (HPA: tissue IHC profile and cell-level ratings).
| Cell-specific reference intensity | Use the named cell population as the comparison unit: HPA rates adrenal glandular cells and lung macrophages High, but adipocytes and skeletal myocytes Not detected (HPA: tissue IHC). A negative neighbouring cell can therefore be informative without making the whole tissue a negative control. |
| Localization and topology | UniProt places ACOT9 in mitochondria, including matrix and inner membrane, and lists no transmembrane segment (UniProt Q9Y305: location and topology). HPA reports a granular mitochondrial pattern in tissue IHC (HPA: tissue IHC profile). Neither source maps the antibody epitope, so these facts do not predict antigen retrieval performance. |
| Isoforms and processing | Four isoforms are listed, and the annotated mature chain spans residues 22–439 (UniProt Q9Y305: isoforms and processing). The supplied record gives no antibody epitope or isoform-specific IHC result; do not assign a distinct staining pattern to an isoform from these annotations alone. |
| IF/ICC Q: Should its localization match tissue IHC? | HPA's ICC-IF summary lists approved plasma membrane and cytosol locations, with additional nucleoplasm, whereas its tissue IHC profile is granular and mitochondrial (HPA: subcellular ICC-IF; tissue IHC). Interpret each application against its own evidence; this IHC section supplies no IF/ICC protocol option. |
| Situation | Likely cause | Next action |
|---|---|---|
| Positive control is blank in adrenal glandular cells or lung macrophages (HPA: High in each). | The intended cell population may be absent, or the staining run may have failed; a blank slide alone does not distinguish these possibilities (general IHC practice). | Confirm the cells on the counterstain, then inspect run controls and each detection step (general IHC practice). Reassess the result only after the control stains as expected. |
| Signal is weak despite identifiable high-staining cells (HPA: tissue IHC ratings). | Low assay signal can reflect the staining workflow (general IHC practice). No ACOT9-specific fixation sensitivity, retrieval condition or working dilution is supplied. | Check the validated IHC-P protocol for the actual antibody, reagent preparation and run controls; optimize retrieval only as a general IHC workflow variable, without assuming an ACOT9-specific effect. |
| Colour is widespread, including blank regions of the section. | Diffuse deposition differs from HPA's granular cytoplasmic profile (HPA: tissue IHC profile); nonspecific detection or endogenous activity may contribute (general IHC practice). | Examine negative and detection-only controls, blocking and reagent application, then score cellular staining only after background is controlled (general IHC practice). |
| Only nuclei or cell borders stain in tissue IHC. | That pattern conflicts with HPA's granular cytoplasmic tissue pattern (HPA: tissue IHC profile), although HPA reports membrane and nucleoplasm in ICC-IF (HPA: subcellular ICC-IF). | Review morphology, controls and staining consistency across expected cell populations before assigning ACOT9 localization; keep the ICC-IF observation separate from the tissue IHC call. |
| Adipocytes or skeletal myocytes stain strongly while expected cells do not. | HPA reports those cell populations Not detected (HPA: adipose tissue; skeletal muscle). Misidentified cells, cross-reactivity or endogenous activity are possible causes (general IHC practice). | Verify cell identity on the counterstain and inspect negative controls; repeat against a section with an identifiable HPA High population before accepting the unexpected pattern. |
| Two sections appear to disagree in overall staining intensity. | Cell composition can shift the apparent tissue-wide signal: HPA ratings apply to specified cells, including High lung macrophages and low hippocampal neurons (HPA: tissue IHC). | Compare matched cell types and their granular cytoplasmic staining, using the same scoring criteria and run controls (general IHC practice); avoid treating tissue-wide darkness as an ACOT9 cell-level rating. |
Experiment evidence
IHC & IF images showing ACOT9 staining
A14147-1 · IHCImmunohistochemistry (IHC) analyzes of ACOT9 pAb bin paraffin-embedded human colon carcinoma tissue at 1:50,showing cytoplasmic staining.Neg
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000123130-ACOT9/tissue/cerebral+cortex#imid_9095888
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000123130-ACOT9/tissue/colon#imid_9095806
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000123130-ACOT9/tissue/liver#imid_9095807
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000123130-ACOT9/tissue/kidney#imid_9095824
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA035533; green = ACOT9, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA035533; green = ACOT9, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ACOT9 IHC & IF
🧪Run bronchus first and expect staining in respiratory epithelial cells (HPA: High in bronchus respiratory epithelial cells); use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes). On the positive slide, assess neighboring stromal cells as potential internal negatives only if they remain unstained; the supplied HPA row does not establish their ACOT9 status (HPA: bronchus respiratory epithelial cells High).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ACOT9 in A-431, U-251MG, U2OS, with annotated localisation: Plasma membrane (approved), Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide; a species- and immunoglobulin-class-matched polyclonal control IgG; and, if available, an ACOT9-knockout biological negative (selected caption: pAb with Goat Anti-Rabbit IgG-biotin secondary). Block endogenous peroxidase and assess endogenous biotin background when using the caption’s avidin–peroxidase detection; for IF, check tissue autofluorescence (selected caption: Goat Anti-Rabbit IgG-biotin followed by avidin-peroxidase).
⚠️Feasibility: An ACOT9-specific fixation window and antigen-retrieval dependency are unreported in the supplied evidence; the selected paraffin-section caption also leaves the fixative unreported (selected SKU A14147-1 caption). That caption shows cytoplasmic staining at 1:50 in paraffin-embedded colon carcinoma, but does not establish whether frozen sections or IF are easier (selected SKU A14147-1 caption). For IF, interpret localization cautiously: HPA reports mainly plasma membrane and cytosol in its ICC-IF images, whereas UniProt places ACOT9 in mitochondria; bronchial tissue autofluorescence may also obscure weak IF signal (HPA: subcellular summary; UniProt Q9Y305 subcellular location).
HPA tissue IHC evidence for ACOT9
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 → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | Synaptic glomeruli - core | High | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | High | Protein (IHC) | HPA → |
Section 3
Advanced ACOT9 IHC Tips
Troubleshoot ACOT9 staining in paraffin sections by checking retrieval, controls, cellular pattern and scoring before interpreting signal (HPA: tissue IHC profile; selected IHC caption: paraffin section).
How should I retrieve ACOT9 in paraffin sections when granular staining is weak?
Use citrate buffer at pH 6.0 for heat-induced epitope retrieval at 95–98 °C for 20 minutes (this page’s retrieval setting). Cool sections before applying the IHC-validated antibody, then compare staining with a section processed in the same run (standard IHC practice). If staining remains weak, adjust retrieval time on adjacent sections while keeping antibody dilution and detection conditions constant (standard IHC practice). Judge improvement by preservation of granular cytoplasmic signal, rather than stronger diffuse staining, and include a primary-antibody omission control to reveal detection background (HPA: tissue IHC profile; selected IHC caption: PBS substituted for primary antibody).
Can I attribute weak ACOT9 staining to the tissue fixative?
Target-specific sensitivity of ACOT9 to fixation is unknown: the selected tissue image identifies a paraffin section but does not report its fixative (selected IHC caption: human colon carcinoma, paraffin embedded; fixative unstated). Record fixative, fixation duration and processing history for each specimen before comparing stain intensity (standard IHC practice). When possible, stain sections with different fixation histories in the same run using the same citrate retrieval at pH 6.0 and 95–98 °C for 20 minutes (this page’s retrieval setting). Interpret a difference cautiously, using section morphology and matched controls to distinguish tissue preservation from staining variation (standard IHC practice).
Should ACOT9 look cytoplasmic, membranous or nuclear in tissue IHC?
Look first for granular cytoplasmic staining in paraffin sections (HPA: cytoplasmic expression with granular, mitochondrial pattern in all tissues). ACOT9 is annotated in the mitochondrial matrix and inner membrane, with no transmembrane segment (UniProt Q9Y305: subcellular location and topology). The selected colon carcinoma image reports cytoplasmic staining at 1:50, without resolving individual mitochondria (selected IHC caption: A14147-1). HPA cell imaging also reports plasma membrane, cytosol and additional nucleoplasm localisation, so document a reproducible alternative pattern without treating it alone as tissue-level proof (HPA: subcellular ICC/IF). Compare cellular detail and omission controls before assigning a nuclear or surface signal to ACOT9 (standard IHC practice).
Could isoforms or epitope processing explain inconsistent ACOT9 staining?
ACOT9 has 4 annotated isoforms, and its annotated mature chain spans residues 22–439 (UniProt Q9Y305: isoforms and processing). Check the catalog antibody’s disclosed immunogen against those sequences before claiming that staining represents every isoform (standard antibody validation practice). An epitope within either annotated HotDog ACOT-type domain, residues 84–209 or 289–401, should be assessed for sequence overlap with the isoforms under study (UniProt Q9Y305: domains; standard antibody validation practice). Lysine 103 is annotated as an acetylation site, but no supplied evidence establishes that this modification changes IHC recognition (UniProt Q9Y305: modified residue; supplied evidence).
How can IF help assess the cellular source of ACOT9 IHC signal?
Use IF as a complementary comparison and multiplex ACOT9 with a marker for the cell population being evaluated, such as a macrophage marker when assessing lung macrophages (HPA: high ACOT9 in lung macrophages; standard IF practice). Choose spectrally separated fluorophores and inspect unstained tissue for autofluorescence before assigning a granular signal to ACOT9 (standard IF practice). Permeabilise sufficiently to expose matrix-facing or other intracellular epitopes if the antibody’s mapped epitope requires access, while checking whether treatment alters cellular detail (UniProt Q9Y305: mitochondrial matrix and inner membrane; standard IF practice). The IHC caption’s 1:50 dilution and paraffin result do not establish an IF dilution or fixation condition (selected IHC caption: A14147-1).
What should I check when ACOT9 chromogen appears throughout the section?
Compare the stained section with a primary-antibody omission control: the selected image used PBS in place of primary antibody and a biotin–avidin–peroxidase detection sequence (selected IHC caption: A14147-1). If both sections develop color, examine peroxidase blocking, endogenous biotin when using avidin–biotin detection, and nonspecific secondary binding (standard IHC practice). Reduce excess primary antibody or detection reagent only after checking that the 1:50 catalog image is comparable to your specimen and workflow (selected IHC caption: A14147-1; standard IHC practice). Retain granular cytoplasmic detail and an appropriate counterstain when adjusting washes or blocking, since HPA describes a granular tissue pattern (HPA: tissue IHC profile; standard IHC practice).
How should I score ACOT9 staining across specimens with different cell mixtures? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because HPA reports a granular cytoplasmic tissue pattern and high staining in selected cell populations (HPA: tissue IHC profile). For chromogenic sections, record the percentage of positive cells and intensity, then calculate an H-score from intensity-weighted percentages using one fixed scoring scheme (standard IHC quantification practice). If counting discrete positive cells, report density per mm² of viable tissue and normalise to the measured area of the same cell population (standard IHC quantification practice). Hold retrieval, detection and image thresholds constant across the comparison, and record omitted-primary background separately (standard IHC practice; selected IHC caption: PBS omission control).
When is an apparent ACOT9-positive focus likely to be artefactual?
A credible tissue signal should be cellular and predominantly granular cytoplasmic, consistent with the reported mitochondrial tissue pattern (HPA: tissue IHC profile). Assess cell identity before comparing foci: HPA reports high staining in lung macrophages and no detected staining in adipocytes, skeletal myocytes or vaginal squamous epithelial cells (HPA: tissue IHC cell-level observations). Treat staining confined to section edges, folds or necrotic areas cautiously, and compare it with intact tissue nearby (standard IHC practice). Color in the omitted-primary section raises concern for endogenous enzyme activity or detection background, while isolated nuclear or surface color needs independent validation before being called ACOT9 in tissue (selected IHC caption: PBS omission control; HPA: tissue IHC profile; standard IHC practice).
Boster reagents
Best ACOT9 / Acyl-coenzyme A thioesterase 9, mitochondrial IHC Antibodies
The catalog antibody has IHC data from a human colon carcinoma paraffin section (A14147-1 IHC caption). Mouse and rat reactivity is listed, but no IF image is supplied (catalog: A14147-1).
Real IHC data
Anti-ACOT9 Antibody
Cat # A14147-1
A14147-1 is listed for IHC and has a human colon carcinoma paraffin-section image showing cytoplasmic staining at 1:50 (catalog applications; A14147-1 IHC caption). Its listed reactivity includes human, mouse and rat; the supplied image documents human tissue only (catalog reactivity; A14147-1 IHC caption).
Which to pick: For tissue IHC, choose A14147-1, a rabbit polyclonal antibody with a paraffin-section IHC image; the caption does not report the fixative (catalog host and applications; A14147-1 IHC caption). No supplied SKU has IF/ICC listed or an IF image (catalog: A14147-1 applications and IF images). For mouse or rat work, A14147-1 lists those species as reactive, although its supplied IHC image shows human tissue only (catalog reactivity; A14147-1 IHC caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9Y305 (ACOT9_HUMAN, Acyl-coenzyme A thioesterase 9, mitochondrial).
- Human Protein Atlas. ACOT9 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. ACOT9 subcellular location (ICC-IF): Mainly localized to the plasma membrane and cytosol. In addition localized to the nucleoplasm..
- Human Protein Atlas. ACOT9 antibody validation summary (1 antibodies).
- ACOT9, a mitochondrial metabolism-related gene, promotes ROS-associated epithelial remodeling in laryngeal squamous cell carcinoma. Journal of translational medicine 2026 — PMC13374139.
- Establishment of prognostic prediction model based on lipid metabolism related genes in esophageal squamous cell carcinoma by machine learning algorithms. BMC gastroenterology 2026 — PMC13335382.
- Prognosis and pain dissection of novel signatures in kidney renal clear cell carcinoma based on fatty acid metabolism-related genes. Frontiers in oncology 2022 — PMC9780486.
- PubMed PMID:10810093 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:17974005 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ACOT9 (Acyl-coenzyme A thioesterase 9, mitochondrial). Expected localisation Granular cytoplasm with a mitochondrial pattern (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A14147-1 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon carcinoma tissue; fixative not specified (datasheet A14147-1); 2. Fixation: Image fixative and duration unreported (datasheet A14147-1); 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-ACOT9, 1:50-1:200 (datasheet A14147-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Adipocytes: not detected; lung macrophages: high (HPA tissue IHC); Isoform / epitope: 4 isoforms; mature chain begins at residue 22 ; epitope effects unknown (UniProt); Regulation: Staining regulation is not established (UniProt). Evidence: UniProt Q9Y305, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the plasma membrane and cytosol. In addition…, Datasheet A14147-1, PMC13374139, PMC13335382. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ACOT9/immunohistochemistry-ihc-guide.