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ACOT8 / Acyl-coenzyme A thioesterase 8 · IHC design guide
Design Immunohistochemistry for ACOT8
Plan chromogenic ACOT8 staining in paraffin sections with the catalog antibody at 1:100–1:300 (datasheet A09392-1). Use liver hepatocytes or kidney tubules as high-staining references, then score cytoplasmic staining and possible granularity (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ACOT8 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic, sometimes granular (HPA tissue IHC) | |
| Staining pattern | Many cell types show cytoplasmic, sometimes granular staining (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 8.0 HIER, heat-mediated (datasheet A09392-1) | |
| 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 fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA show only medium consistency (HPA tissue IHC) | |
| Regulation | Low tissue specificity (HPA tissue RNA) | |
| Isoform / epitope | No annotated isoforms; one 1–319 chain (UniProt) |
Section 1
Recommended ACOT8 IHC & IF Protocols
Start with the catalog antibody’s IHC-P protocol (datasheet A09392-1). A published breast cancer tissue microarray protocol offers another workflow (PMC11346483).
| Sample | Paraffin-embedded Human brain tissue; fixative not specified (datasheet A09392-1) |
| Fixation | Image fixative and duration unreported (datasheet A09392-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Tris-EDTA pH 8.0 (datasheet A09392-1); 20 min, 95–100 °C (standard) |
| 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-ACOT8, 1:100-1:300 (datasheet A09392-1) |
| 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 | ACOT8-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression, sometimes in a granular pattern. No signal in the no-primary control. |
💡Decision noteTry heat-mediated Tris-EDTA pH 8.0 retrieval first (datasheet A09392-1); compare EDTA pH 9 if needed (PMC11346483).
Section 2
What Is the Expected ACOT8 Staining Pattern?
ACOT8 is predominantly a peroxisome-matrix protein, with possible cytosolic localization and no transmembrane segment (UniProt O14734). In paraffin-section IHC, expect cytoplasmic staining, sometimes granular, across many tissues; HPA reports high staining in hepatocytes and kidney tubule cells, among others (HPA: tissue IHC). The HPA tissue profile is Approved, with medium consistency between staining and RNA expression (HPA: tissue IHC).
| Cytoplasmic staining, sometimes granular, in hepatocytes or kidney tubule cells (HPA: tissue IHC). | This fits the reported IHC pattern in two high-staining cell populations (HPA: tissue IHC). Peroxisome-matrix localization supports a cytoplasmic pattern, but chromogenic granules alone do not identify individual peroxisomes (UniProt O14734; general IHC practice). |
| Predominantly nuclear staining, with little cytoplasmic signal. | A nuclear-dominant pattern conflicts with the reported cytoplasmic IHC profile and peroxisome-matrix localization (HPA: tissue IHC; UniProt O14734). Treat it as suspect and review the counterstain, detection background, and antibody controls before scoring it as ACOT8 (general IHC practice). |
| Strong staining confined to a cell population outside the reported high-staining populations (HPA: tissue IHC). | Check cell identity and compare a known-positive area; cross-reactivity or endogenous detection activity is possible (general IHC practice). Staining outside the listed examples is not automatically false: HPA describes ubiquitous expression and supplies no negative tissue (HPA: tissue IHC). |
| Uniform haze over tissue and empty areas, obscuring cell boundaries. | A field-wide haze is less interpretable than the reported cytoplasmic pattern (HPA: tissue IHC). Consider nonspecific antibody binding, incomplete blocking, or detection background; assess a control without primary antibody and review washing and reagent concentration (general IHC practice). |
| No convincing signal in hepatocytes or kidney tubule cells. | Both are reported as high-staining IHC populations, so a blank result warrants a workflow check (HPA: tissue IHC). Verify tissue identity, antibody and detection performance, retrieval conditions, and a known-positive control before calling the sample negative (general IHC practice). |
💡Expected ACOT8 appearanceCall a positive result when cytoplasmic, sometimes granular staining appears in reported high-staining cells such as hepatocytes or kidney tubule cells (HPA: tissue IHC); nuclear-dominant staining or field-wide haze is suspect (HPA: tissue IHC; general IHC practice).
| Tissue and cell selection | HPA reports high staining in liver hepatocytes, kidney tubule cells, and several glandular populations; vagina squamous epithelium, smooth muscle cells, and bone marrow hematopoietic cells are low (HPA: tissue IHC). Use a reported high population as a positive reference, without treating a low population as a true negative. |
| Compartment and image interpretation | UniProt places ACOT8 predominantly in the peroxisome matrix but cannot exclude cytosol (UniProt O14734). HPA describes ubiquitous cytoplasmic IHC staining, sometimes granular (HPA: tissue IHC). Score the visible cytoplasmic pattern; granularity alone cannot prove organelle identity (general IHC practice). |
| Antibody evidence | The listed rabbit polyclonal antibody CAB010261 is Approved for IHC; the tissue profile has medium staining-to-RNA consistency (HPA: antibodies; HPA: tissue IHC). Interpret unexpected patterns with controls rather than assuming that approval establishes specificity in every specimen (general IHC practice). |
| Molecular features and retrieval | UniProt annotates one chain of 319 amino acids, no transmembrane segment, no signal peptide, no propeptide, and no isoforms (UniProt O14734). These annotations do not establish an ACOT8-specific antigen-retrieval setting or fixation sensitivity; optimize retrieval using a positive-control section (general IHC practice). |
| IF/ICC cross-check: should puncta be expected? | HPA ICC-IF reports vesicles as the supported main location and mitochondria as an approved additional location (HPA: subcellular). That can inform an IF/ICC comparison, but it does not redefine the reported cytoplasmic, sometimes granular pattern in paraffin-section IHC (HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| A reported high-staining control is blank (HPA: tissue IHC). | The antibody, retrieval, or chromogenic detection step may have failed (general IHC practice). | Confirm the tissue and cell population, run a known-positive section, then check antibody preparation, retrieval conditions, and detection reagents one step at a time (general IHC practice). |
| Signal is predominantly nuclear. | This conflicts with the cytoplasmic IHC profile and peroxisome-matrix annotation (HPA: tissue IHC; UniProt O14734). | Check counterstain interpretation and compare sections with and without primary antibody; reassess specificity before assigning nuclear signal to ACOT8 (general IHC practice). |
| Staining covers tissue and empty slide areas. | Diffuse detection or nonspecific binding can obscure cell-associated cytoplasmic signal (general IHC practice; HPA: tissue IHC). | Inspect the control without primary antibody; review blocking, washing, antibody concentration, and chromogen development using standard IHC controls (general IHC practice). |
| Unexpected cells stain strongly. | Cell identity, cross-reactivity, or endogenous detection activity may explain the result (general IHC practice). HPA also reports ubiquitous expression, so an unlisted cell is not inherently negative (HPA: tissue IHC). | Confirm the cells on the counterstained section, compare a reported high-staining population, and use detection controls before assigning the signal to ACOT8 (general IHC practice; HPA: tissue IHC). |
| Only weak, patchy cytoplasmic staining appears in a reported high population (HPA: tissue IHC). | Section-to-section variation or a suboptimal IHC workflow is possible; HPA rates staining-to-RNA consistency as medium (general IHC practice; HPA: tissue IHC). | Compare a positive-control section processed in the same run and review retrieval, antibody incubation, and chromogen development before interpreting intensity differences (general IHC practice). |
| Granules are visible, but their identity is uncertain. | Granular cytoplasmic IHC is reported, while chromogenic morphology alone cannot establish peroxisomal identity (HPA: tissue IHC; general IHC practice). | Record the cytoplasmic and granular pattern without labeling individual granules as peroxisomes; use the separate IF/ICC guide if organelle-level localization is the question (HPA: tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing ACOT8 staining
A09392-1 · IHCImmunohistochemical analysis of paraffin-embedded Human brain. Antibody was diluted at 1:100 (4° overnight). High-pressure and temperature T
A09392-1 · IFImmunofluorescence analysis of A549 cells, using ACOT8 Antibody. The picture on the right is blocked with the synthesized peptide.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000101473-ACOT8/tissue/cerebral+cortex#imid_2869380
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000101473-ACOT8/tissue/colon#imid_2869314
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000101473-ACOT8/tissue/liver#imid_2869315
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000101473-ACOT8/tissue/kidney#imid_2869308
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB010261; green = ACOT8, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB010261; green = ACOT8, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ACOT8 IHC & IF
🧪Run cerebellum first and assess staining in Purkinje cells (HPA: High in cerebellar Purkinje cells). HPA detects ACOT8 in all 44 scored tissues, so there is no HPA-supported negative tissue; use no-primary and isotype controls, and treat adjacent cells with little or no signal as within-slide background comparisons rather than confirmed ACOT8-negative cells (HPA: no negative tissue rows).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: ACOT8 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 ACOT8 in A-431, U-251MG, U2OS, with annotated localisation: Vesicles (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide and a control matched to the primary antibody’s host and class (isotype IgG for a monoclonal antibody, or normal host IgG for a polyclonal antibody); use ACOT8 knockout material or immunogen-peptide preabsorption as a biological specificity control (selected SKU A09392-1 tissue-IHC caption: peptide-preabsorbed control). For chromogenic IHC, quench endogenous peroxidase; for cerebellar IF, assess neuronal lipofuscin autofluorescence in an unstained control (standard IHC/IF practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the fixative is unreported in the selected SKU A09392-1 paraffin-section brain caption (selected SKU A09392-1 tissue-IHC caption). That caption used high-pressure, high-temperature Tris-EDTA retrieval at pH 8.0, but it does not establish that ACOT8 staining depends on that retrieval method (selected SKU A09392-1 tissue-IHC caption). The supplied evidence does not establish whether frozen sections or IF are easier than paraffin IHC; cerebellar neuronal lipofuscin can complicate IF interpretation (standard IF practice).
HPA tissue IHC evidence for ACOT8
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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 → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: ACOT8 is detected in all 44 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced ACOT8 IHC Tips
Troubleshoot ACOT8 staining in paraffin section IHC using the catalog image, tissue expression data, and compartment checks; use IF as a complementary localisation assay.
Which retrieval conditions should I start with for ACOT8 in paraffin sections?
Start with heat mediated Tris-EDTA antigen retrieval at pH 8.0 (datasheet A09392-1). The catalog image used high pressure and temperature retrieval in paraffin embedded human brain, followed by antibody at 1:100 overnight at 4°C (caption A09392-1). Keep heating, cooling, and section handling consistent between runs, then compare staining with a matched negative control (standard IHC practice). If staining is weak, adjust heating duration cautiously before considering another retrieval buffer as a fallback, because excessive heating can damage morphology (standard IHC practice). Record the conditions used so changes in signal can be assessed against the same tissue and control (standard IHC practice).
How should I investigate weak ACOT8 staining when fixation history varies?
Target specific sensitivity of ACOT8 to fixation is unknown from the supplied evidence; the catalog brain caption identifies paraffin embedding but does not state a fixative (caption A09392-1). Record fixative, fixation duration, tissue thickness, and processing history for each section before comparing signal (standard IHC practice). Run sections with different histories in the same staining batch, using the same pH 8.0 Tris-EDTA retrieval and antibody conditions where appropriate (datasheet A09392-1; standard IHC practice). Compare morphology and cytoplasmic signal in comparable cell populations, and include a negative control for each processing group (HPA tissue IHC; standard IHC practice). Do not assign a fixation effect from staining differences alone (standard IHC practice).
Does diffuse ACOT8 staining conflict with its expected peroxisomal localisation?
Assess staining as cytoplasmic first: tissue IHC is described as ubiquitous cytoplasmic expression, sometimes granular (HPA tissue IHC). ACOT8 is predominantly assigned to the peroxisome matrix, although cytosolic localisation cannot be excluded (UniProt O14734 subcellular location). Fine granules may be difficult to resolve in chromogenic sections, so compare their distribution across cells at the same section thickness and detection settings (standard IHC practice). Diffuse cytoplasm alone therefore does not settle whether the signal is peroxisomal or cytosolic (UniProt O14734 subcellular location; standard IHC practice). If compartment assignment matters, compare serial IHC sections with a validated peroxisome marker or use colocalisation by IF (standard IHC / IF practice).
How can I assess epitope access when the ACOT8 antibody binding site is unspecified?
The supplied record lists a 319 amino acid chain, no annotated isoforms, no transmembrane segment, and no annotated glycosylation sites or modified residues (UniProt O14734 record). The binding epitope of A09392-1 is not supplied, so those annotations cannot establish whether retrieval exposes its epitope (datasheet A09392-1; UniProt O14734 record). Begin with the documented pH 8.0 Tris-EDTA retrieval and compare signal with the catalog antibody preabsorbed by immunogen peptide when that control is available (caption A09392-1). Evaluate preservation of cell morphology alongside staining intensity, since harsh retrieval can alter tissue appearance (standard IHC practice). If comparing antibodies, document each antibody's stated epitope before treating staining differences as evidence of an ACOT8 isoform (standard IHC practice; UniProt O14734 record).
How can IF help resolve ACOT8 localisation seen in chromogenic IHC?
Use IF as a complementary compartment check: tissue IHC reports cytoplasmic staining that can be granular, while the subcellular IF summary places ACOT8 mainly in vesicles and additionally in mitochondria (HPA tissue IHC; HPA subcellular). Multiplex ACOT8 with a marker for the expected cell type and, when testing compartment identity, a validated peroxisome marker (HPA tissue IHC; UniProt O14734 subcellular location; standard IF practice). Choose separated fluorophores and inspect unstained tissue to place the weaker signal away from prominent autofluorescence (standard IF practice). Because ACOT8 is assigned to the peroxisome matrix and lacks a transmembrane segment, permeabilisation must provide access to the intracellular epitope, whose exact position for this antibody is unreported (UniProt O14734 record; standard IF practice).
What controls distinguish ACOT8 staining from chromogenic background?
Include a section without primary antibody to reveal background from the detection system, and compare it with a stained section processed in the same run (standard IHC practice). The catalog brain image includes a negative control made by preabsorbing the antibody with immunogen peptide; use that comparison when the peptide is available (caption A09392-1). Apply a peroxidase block before peroxidase based chromogenic detection and assess whether residual pigment or endogenous activity resembles the ACOT8 pattern (standard IHC practice). Titrate antibody and detection exposure if signal spreads into acellular areas or obscures cell boundaries (standard IHC practice). Judge residual staining against the expected cytoplasmic, sometimes granular, tissue pattern (HPA tissue IHC).
How should I quantify ACOT8 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, then use either an H-score combining positive cell fraction and intensity or report the percentage of positive cells (standard IHC practice). For spatial questions, count positive cells per mm² of viable tissue rather than per whole image (standard IHC practice). Normalise comparisons to the same cell type, viable area, section processing, and imaging or scoring settings (standard IHC practice). Report cytoplasmic intensity separately from any granular pattern so changes in distribution are visible (HPA tissue IHC; standard IHC practice). Include matched controls and avoid equating chromogen intensity directly with enzyme activity (UniProt O14734 function; standard IHC practice).
Which patterns support genuine ACOT8 staining rather than artefact?
Look for reproducible cytoplasmic staining in intact cells, with possible granularity, rather than interpreting an isolated dark deposit as ACOT8 (HPA tissue IHC; standard IHC practice). Hepatocytes, kidney tubule cells, and cerebellar Purkinje cells are reported as high staining examples, while smooth muscle cells are reported as low staining (HPA tissue IHC). Treat dominant nuclear or extracellular signal, section edge enhancement, and staining confined to necrotic areas as reasons to inspect controls and tissue quality (UniProt O14734 subcellular location; standard IHC practice). Check whether a no-primary control reveals endogenous enzyme signal, and compare peptide preabsorption when available (standard IHC practice; caption A09392-1). Tissue IHC evidence has medium consistency with RNA expression, so interpret isolated discrepancies cautiously (HPA tissue IHC).
Boster reagents
Best ACOT8 / Acyl-coenzyme A thioesterase 8 IHC Antibodies
The anti-ACOT8 catalog antibody has IHC data from paraffin-embedded human brain and IF data from A549 cells; listed reactivity covers human, mouse, and rat (IHC/IF captions; catalog reactivity).
Real IHC data
Anti-Acyl-coenzyme A thioesterase 8 ACOT8 Antibody
Cat # A09392-1
A09392-1 is listed for IHC and IF, with an IHC image from paraffin-embedded human brain (catalog applications; IHC caption). Its IF image uses A549 cells, and its listed reactivity includes human, mouse, and rat (IF caption; catalog reactivity).
Which to pick: For tissue IHC, choose A09392-1: its own image shows paraffin-embedded human brain stained at 1:100 after Tris-EDTA retrieval at pH 8.0; the fixative is unreported (IHC caption). For IF/ICC, A09392-1 has IF data in A549 cells with a peptide-blocked comparator (IF caption). For cross-species work, A09392-1 is a rabbit polyclonal listed as reactive with human, mouse, and rat, although its provided IHC image documents human tissue only (catalog host/clonality/reactivity; IHC caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O14734 (ACOT8_HUMAN, Acyl-coenzyme A thioesterase 8).
- Human Protein Atlas. ACOT8 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ACOT8 subcellular location (ICC-IF): Mainly localized to vesicles. In addition localized to the mitochondria..
- Human Protein Atlas. ACOT8 antibody validation summary (1 antibodies).
- Acyl-CoA Thioesterase 8 (ACOT8) is a Poor Prognostic Biomarker in Breast Cancer. Pharmacogenomics and personalized medicine 2024 — PMC11346483.
- Acyl-CoA Thioesterase 8 and 11 as Novel Biomarkers for Clear Cell Renal Cell Carcinoma. Frontiers in genetics 2020 — PMC7758486.
- Hepatic Lamp2a deficiency promotes inflammation of murine autoimmune cholangitis via affecting bile acid metabolism. iScience 2025 — PMC11849667.
- PubMed PMID:9299485 — UniProt-cited evidence.
- PubMed PMID:9153233 — UniProt-cited evidence.
- PubMed PMID:10092594 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ACOT8 (Acyl-coenzyme A thioesterase 8). Expected localisation Cytoplasmic, sometimes granular (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 8.0 HIER, heat-mediated (datasheet A09392-1). Validated IHC antibody: A09392-1 at 1:100-1:300 (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 workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded Human brain tissue; fixative not specified (datasheet A09392-1); 2. Fixation: Image fixative and duration unreported (datasheet A09392-1); 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 retrieval: Tris-EDTA pH 8.0 (datasheet A09392-1); 20 min, 95–100 °C (standard); 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-ACOT8, 1:100-1:300 (datasheet A09392-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Staining and RNA show only medium consistency (HPA tissue IHC); Isoform / epitope: No annotated isoforms; one 1–319 chain (UniProt); Regulation: Low tissue specificity (HPA tissue RNA). Evidence: UniProt O14734, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to vesicles. In addition localized to the…, Datasheet A09392-1, PMC11346483. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ACOT8/immunohistochemistry-ihc-guide.