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ACOT8 / Acyl-coenzyme A thioesterase 8 · IHC design guide
Design Immunohistochemistry for ACOT8
Plan ACOT8 chromogenic IHC in paraffin sections with a starting antibody dilution of 1:100–1:300 (datasheet A09392-1). Use liver hepatocytes or kidney tubules as high-staining reference cells (HPA tissue IHC), and assess cytoplasmic staining that may appear granular (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 | Observed cytoplasm; peroxisome matrix expected (HPA tissue IHC; UniProt) | |
| Staining pattern | Cytoplasmic staining, sometimes granular, across tissues (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 paraffin-section fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A09392-1) | |
| Caveat | Cytosolic signal may accompany peroxisomal signal (UniProt) | |
| Regulation | Ubiquitous expression (UniProt) | |
| Isoform / epitope | No annotated isoforms or signal/propeptide; epitope unknown (UniProt) |
Section 1
Recommended ACOT8 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: A09392-1) is supplemented by one published ACOT8 protocol for breast cancer tissue (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 noteStart with heat-mediated Tris-EDTA pH 8.0 retrieval (datasheet: A09392-1); EDTA pH 9 is a published alternative (PMC11346483).
Section 2
What Is the Expected ACOT8 Staining Pattern?
ACOT8 is predominantly in the peroxisome matrix, although cytosolic localization cannot be excluded; it has no transmembrane segment (UniProt O14734: localization and topology). In paraffin-section IHC, expect cytoplasmic staining that may look granular, especially in hepatocytes, kidney tubular cells, and several glandular cell populations (HPA: tissue IHC). HPA rates the tissue staining Approved, with medium consistency between staining and RNA expression (HPA: tissue IHC reliability).
| Cytoplasmic staining, sometimes granular, is strong in hepatocytes or kidney tubular cells. | This fits the reported IHC pattern: both cell populations are rated High, and the overall profile is ubiquitous cytoplasmic expression that can be granular (HPA: tissue IHC). Granules are compatible with the predominantly peroxisomal localization, but chromogenic appearance alone does not establish peroxisome identity (UniProt O14734: localization; general IHC interpretation). |
| The nucleus is the dominant stained compartment while cytoplasm is faint or unstained. | A predominantly nuclear result does not match the reported cytoplasmic tissue pattern or peroxisome-matrix localization (HPA: tissue IHC; UniProt O14734: localization). Treat it as a possible staining artefact and compare with an appropriate control and the expected positive cells before assigning biological meaning (general IHC practice). |
| Hematopoietic cells dominate the signal while nearby expected positive cells stain weakly. | HPA rates bone-marrow hematopoietic cells Low, while hepatocytes and kidney tubular cells are High (HPA: tissue IHC). A reversal of that expected pattern warrants checks for cross-reactivity or endogenous detection activity; Low means reduced reported staining, not proven absence of ACOT8 (HPA: tissue IHC; general IHC practice). |
| Color covers many cells or the section broadly, obscuring cytoplasmic boundaries. | Diffuse background prevents a reliable call about ACOT8 localization or cell-specific intensity (general IHC interpretation). Inspect a matched negative control for nonspecific antibody binding or detection background, then reassess whether discrete cytoplasmic signal remains in expected positive cells (general IHC practice; HPA: tissue IHC). |
| A liver or kidney section shows no convincing cytoplasmic signal. | Hepatocytes and kidney tubular cells are reported High, so a blank result in either is unexpected under a working assay (HPA: tissue IHC). First check section quality, reagent performance, retrieval conditions, and detection controls as general IHC variables; the supplied sources do not establish ACOT8-specific fixation or retrieval sensitivity (general IHC practice). |
💡Expected ACOT8 appearanceCall a section positive when expected cells show clear cytoplasmic, sometimes granular staining—especially High-rated hepatocytes or kidney tubular cells; dominant nuclear color or indiscriminate background is suspect (HPA: tissue IHC; UniProt O14734: localization; general IHC interpretation).
| Compartment and morphology | The peroxisome matrix is the predominant UniProt location, with cytosol not excluded; HPA describes ubiquitous cytoplasmic IHC that can be granular (UniProt O14734: localization; HPA: tissue IHC). Neither source makes every cytoplasmic granule a confirmed peroxisome. |
| Choice of comparison cells | HPA rates hepatocytes, kidney tubular cells, Purkinje cells, and listed glandular populations High; squamous epithelial cells of the vagina, smooth muscle cells, and bone-marrow hematopoietic cells are Low (HPA: tissue IHC). These are relative staining references, not absolute positive and negative controls. |
| Strength of tissue evidence | The HPA tissue profile is Approved with medium consistency between antibody staining and RNA expression; CAB010261 is IHC Approved (HPA: tissue IHC reliability; HPA: antibodies). Interpret an unusual pattern with controls rather than treating the profile as a guarantee for every section (general IHC practice). |
| What should IF/ICC show? | HPA reports mainly vesicular localization, with additional mitochondrial localization, in ICC-IF; its vesicle location is Supported and mitochondrial location Approved (HPA: subcellular ICC-IF). UniProt places ACOT8 predominantly in the peroxisome matrix and does not exclude cytosol (UniProt O14734: localization). Resolve a compartment claim with suitable markers rather than appearance alone (general IF practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| No staining in liver hepatocytes or kidney tubular cells. | Both are High in the HPA tissue profile, so assay failure or unsuitable staining conditions are possible (HPA: tissue IHC; general IHC practice). | Check the section and detection controls, then review retrieval and antibody incubation under the chosen general IHC workflow; no ACOT8-specific fixation or retrieval effect is supplied (general IHC practice). |
| Strong signal appears mainly in nuclei. | This conflicts with the reported cytoplasmic IHC pattern and peroxisome-matrix localization; artefact is possible (HPA: tissue IHC; UniProt O14734: localization). | Compare expected positive cells with a matched negative control, then reassess the detection signal and staining specificity (general IHC practice). |
| Color appears without a clear cellular pattern. | Nonspecific background or endogenous detection activity can obscure specific chromogenic staining (general IHC practice). | Check a control lacking primary antibody and review blocking, washing, and detection controls before interpreting tissue distribution (general IHC practice). |
| Bone-marrow hematopoietic cells stain more strongly than the expected positive cells. | HPA rates these cells Low, so a strong reversal raises concern about background or cross-reactivity; Low does not mean absent (HPA: tissue IHC; general IHC interpretation). | Compare the same run with a High-rated tissue and a matched negative control; assess the cellular distribution before scoring ACOT8 (HPA: tissue IHC; general IHC practice). |
| Only weak, diffuse cytoplasm is visible in a High-rated tissue. | HPA permits cytoplasmic staining without obvious granules, but weak signal can also reflect a technical limitation (HPA: tissue IHC; general IHC practice). | Judge signal against background and an expected positive control; do not require visible puncta as the sole criterion for positivity (HPA: tissue IHC; general IHC interpretation). |
| A granular pattern is interpreted as definitive peroxisomal staining. | Granularity is reported in tissue IHC, but morphology alone cannot identify the organelle; ICC-IF also reports vesicles and mitochondria (HPA: tissue IHC; HPA: subcellular ICC-IF). | Describe the IHC result as granular cytoplasmic staining. Reserve organelle assignment for an appropriately controlled localization experiment (general IHC/IF interpretation). |
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
🧪Start with cerebellum and assess cytoplasmic staining in Purkinje cells (HPA: High in cerebellar Purkinje cells). HPA detects ACOT8 in all 44 scored tissues, so no negative tissue is available; use no-primary and isotype controls to define background, and treat neighboring cells showing counterstain alone as internal background comparators, not validated ACOT8-negative cells (HPA: detected in all 44 scored tissues).
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: Run a no-primary (secondary-only) control, an isotype control matched to the primary antibody’s host species and monoclonal subclass if applicable, and a peptide-blocked primary control (selected SKU A09392-1 caption: immunogen-peptide preabsorption). Quench endogenous peroxidase for chromogenic IHC; in cerebellum, distinguish pigment from chromogen and check lipofuscin autofluorescence if using IF (standard IHC/IF practice).
⚠️Feasibility: An ACOT8-specific fixation window or fixation effect is unreported, and the selected A09392-1 paraffin-brain caption does not state a fixative (selected SKU A09392-1 caption). The caption uses high-pressure, high-temperature Tris-EDTA retrieval at pH 8.0 and 1:100 antibody overnight at 4°C; it provides a starting condition but does not establish that retrieval is required (selected SKU A09392-1 caption). Frozen-section and tissue-IF performance are unreported, so neither can be judged easier; Purkinje-cell lipofuscin may complicate pigment or fluorescence interpretation (standard IHC/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
Use the catalog antibody’s paraffin-section evidence alongside ACOT8 localisation and tissue-expression data when troubleshooting chromogenic IHC.
Which retrieval conditions should I try first for weak ACOT8 staining?
Start with heat-mediated retrieval in Tris-EDTA at pH 8.0 (datasheet A09392-1). The paraffin-section brain image used high pressure and temperature with that buffer, followed by antibody at 1:100 overnight at 4°C (caption A09392-1). If staining is weak, check whether sections were fully dewaxed and whether retrieval temperature reached the tissue evenly before changing the buffer (standard IHC practice). Compare changes on adjacent sections of the same specimen, keeping antibody incubation and chromogen development constant so retrieval effects remain interpretable (standard IHC practice).
Could fixation explain loss of ACOT8 signal in my paraffin sections?
The selected paraffin-section caption does not state a fixative, so target-specific sensitivity to fixation is unknown (caption A09392-1). Record the fixative, fixation duration and processing history for each specimen before comparing ACOT8 staining across cases (standard IHC practice). In a troubleshooting run, process matched samples consistently and compare them with the same Tris-EDTA pH 8.0 retrieval and antibody incubation (datasheet A09392-1; standard IHC practice). If signal differs, evaluate tissue preservation and overall staining quality alongside ACOT8; the difference alone cannot establish a particular fixation effect on this antigen (standard IHC practice).
What ACOT8 staining pattern should I expect in tissue?
Assess ACOT8 within the cytoplasm, including a possible granular pattern, because that is the reported tissue-IHC profile (HPA tissue IHC: ubiquitous cytoplasmic expression, sometimes granular). A punctate signal is biologically plausible because ACOT8 is predominantly assigned to the peroxisome matrix, although cytosolic localisation cannot be excluded (UniProt O14734 subcellular location). Do not require every positive cell to show individually resolved puncta in chromogenic sections; section thickness, chromogen spread and microscope resolution affect their appearance (standard IHC practice). Score cytoplasmic staining in identified cells and document diffuse versus granular patterns separately, especially when comparing specimens (standard IHC practice).
How should I investigate inconsistent staining across ACOT8 antibodies?
Check each antibody’s stated immunogen or epitope and its validation in paraffin sections before treating discordant staining as biology (standard IHC practice). The supplied record annotates one 319-aa chain, no isoforms, and no signal peptide or propeptide; it does not establish where this antibody binds (UniProt O14734 processing and isoforms). ACOT8 has no annotated transmembrane segment, glycosylation sites or modified residues in this record, so those features provide no documented explanation for antibody differences (UniProt O14734 topology and PTMs). Compare staining in adjacent sections under each antibody’s validated retrieval conditions and investigate discordance with an independent specificity control (standard IHC practice).
How can IF help resolve the cellular source of ACOT8 staining?
For a separate IF experiment, pair ACOT8 with a marker for the cell type being examined, such as a hepatocyte marker in liver, where hepatocytes show high tissue-IHC staining (HPA tissue IHC: high in hepatocytes). Choose fluorophores whose emission can be distinguished from tissue autofluorescence, and include single-channel controls when assessing overlap (standard IF practice). After fixation, optimise permeabilisation for access to an intracellular epitope, accounting for ACOT8’s predominantly peroxisome-matrix assignment and possible cytosolic localisation (UniProt O14734 subcellular location; standard IF practice). Treat the reported vesicle and mitochondrial IF locations as additional observations to investigate, rather than assuming every IF punctum is peroxisomal (HPA subcellular).
How do I distinguish ACOT8 staining from chromogenic background?
Include a no-primary control to reveal signal from detection reagents, and assess endogenous peroxidase blocking when using a peroxidase and DAB workflow (standard IHC practice). The selected brain-section image also reports a peptide-preabsorbed negative control, which is relevant to that antibody and image (caption A09392-1). Compare background in tissue spaces, section edges and damaged regions with staining inside intact cells; widespread deposits outside cellular structures weaken the case for specific ACOT8 signal (standard IHC practice). Optimise blocking, antibody concentration and wash conditions systematically, changing one variable at a time while retaining the documented 1:100 condition as a reference (caption A09392-1; standard IHC practice).
What is a defensible way to score ACOT8 IHC? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, since the reported tissue pattern is cytoplasmic and sometimes granular (HPA tissue IHC: cytoplasmic expression). Within comparable regions, report the percentage of positive cells and an intensity-weighted H-score, or quantify positive-cell density per mm² when cell counts are the main endpoint (standard IHC practice). Normalise scores to the number of evaluable cells or tissue area in that same annotated compartment, excluding folds, necrosis and empty spaces (standard IHC practice). Keep section thickness, imaging, staining development and scoring thresholds consistent, and report diffuse and granular cytoplasmic staining separately if both occur (standard IHC practice).
Which findings would make an apparent ACOT8-positive result questionable?
A credible result should include intracellular cytoplasmic staining in intact cells, potentially granular, consistent with the reported tissue profile (HPA tissue IHC: cytoplasmic expression, sometimes granular). High staining in liver hepatocytes or kidney tubular cells can provide tissue context, but expression levels alone do not prove antibody specificity (HPA tissue IHC: high in hepatocytes and kidney tubular cells; standard IHC practice). Treat dominant nuclear signal, staining confined to cut edges or necrosis, and deposits reproduced in a no-primary control as reasons to investigate artefact (UniProt O14734 subcellular location; standard IHC practice). For peroxidase detection, also check endogenous enzyme activity before attributing DAB deposits to ACOT8 (standard IHC practice).
Boster reagents
Best ACOT8 / Acyl-coenzyme A thioesterase 8 IHC Antibodies
A09392-1 has IHC data from paraffin-embedded human brain (IHC image caption) and IF data from A549 cells (IF image caption). Its listed reactivity covers human, mouse and rat (catalog reactivity).
Real IHC data
Anti-Acyl-coenzyme A thioesterase 8 ACOT8 Antibody
Cat # A09392-1
A09392-1 is shown staining paraffin-embedded human brain by IHC (IHC image caption). A09392-1 is also shown in IF images of A549 cells, including a peptide-blocked comparison (IF image caption).
Which to pick: Choose A09392-1 for paraffin-section tissue IHC: its own human-brain image used 1:100 antibody overnight at 4°C with Tris-EDTA pH 8.0 retrieval; the fixative is unreported (IHC image caption). For IF/ICC, A09392-1 is the listed option because the rabbit polyclonal antibody lists IF and has an A549-cell IF image (catalog host and dilution data; catalog applications; IF image caption). For mouse or rat work, A09392-1 lists reactivity with both species, while the supplied IHC and IF images show human brain and A549 cells, respectively (catalog reactivity; IHC image caption; IF image 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 Observed cytoplasm; peroxisome matrix expected (HPA tissue IHC; UniProt). 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 paraffin-section fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A09392-1); Caveat: Cytosolic signal may accompany peroxisomal signal (UniProt); Isoform / epitope: No annotated isoforms or signal/propeptide; epitope unknown (UniProt); Regulation: Ubiquitous expression (UniProt). 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.