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ACAA2 / 3-ketoacyl-CoA thiolase, mitochondrial · IHC design guide
Design Immunohistochemistry for ACAA2
Start paraffin-section IHC with the catalog antibody at 1:50 (datasheet M08341-1). Compare granular cytoplasmic staining in hepatocytes with adipocytes, while accounting for low agreement between antibody staining and RNA expression (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ACAA2 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 observed (HPA tissue IHC); mitochondrion annotated (UniProt) | |
| Staining pattern | Granular cytoplasm in hepatocytes and glandular cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M08341-1) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Adipose tissue+4 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) | |
| Caveat | Antibody staining has low concordance with RNA (HPA tissue IHC) | |
| Regulation | No expression regulator annotated (UniProt) | |
| Isoform / epitope | No annotated isoforms; no isoform-dependent epitope map (UniProt) |
Section 1
Recommended ACAA2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet M08341-1) is paired with published ACAA2 IHC methods (PMC12771824; PMC11940541).
| Sample | Paraffin-embedded human colorectal adenocarcinoma tissue; fixative not specified (datasheet M08341-1) |
| Fixation | Image fixative and duration unreported (datasheet M08341-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet M08341-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M08341-1) |
| Primary antibody | Rabbit monoclonal (clone 26A90) anti-ACAA2, 1:50 (datasheet M08341-1) |
| Primary incubation | Overnight at 4 °C (datasheet M08341-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet M08341-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ACAA2-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression with granular pattern in several tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet M08341-1). Citrate at pH 6.0 is a published alternative (PMC12771824 methods).
Section 2
What Is the Expected ACAA2 Staining Pattern?
ACAA2 is mitochondrial and has no annotated transmembrane segment (UniProt P42765 topology). In paraffin sections, expect granular cytoplasmic staining, especially in hepatocytes, kidney tubule cells, and intestinal glandular cells (HPA: tissue IHC). Interpret these examples with care: HPA rates the tissue profile Approved but reports low agreement with RNA data and pending external verification (HPA: tissue reliability).
| Distinct granular cytoplasmic staining in hepatocytes or kidney tubule cells. | This fits the reported tissue pattern and strong staining in those cells (HPA: tissue IHC). Granules are consistent with mitochondrial ACAA2 (UniProt P42765 location); chromogenic IHC alone does not prove organelle identity. |
| Predominantly nuclear, membrane, or extracellular staining. | These compartments conflict with the main mitochondrial assignment (UniProt P42765; HPA: subcellular ICC-IF). Consider nonspecific signal or a detection artefact; assess whether expected cytoplasmic granules are present in the same section. |
| Strong staining in adipocytes or skeletal myocytes. | Both are reported as not detected by HPA tissue IHC. Treat strong signal there as unexpected and investigate cross-reactivity or endogenous detection activity before interpreting it as ACAA2 (HPA: tissue IHC). |
| Broad, smooth colour across cells, stroma, and empty spaces. | This differs from HPA’s granular cytoplasmic pattern (HPA: tissue IHC). General IHC practice: inspect a no-primary control and review blocking, washing, and chromogen development for background signal. |
| No stain in hepatocytes or kidney tubule cells. | Both are reported as High and can serve as positive tissue checks (HPA: tissue IHC). First verify that the run detected its positive control; absent signal alone cannot establish that the test specimen lacks ACAA2. |
💡Expected ACAA2 appearanceCall positive when strong, granular cytoplasmic staining appears in hepatocytes, kidney tubule cells, or the listed intestinal glandular cells (HPA: tissue IHC), consistent with mitochondrial localization (UniProt P42765); dominant nuclear staining or equally strong signal in HPA not-detected cell types warrants investigation.
| Which cells provide useful tissue contrasts? | Hepatocytes and kidney tubule cells are High; adipocytes and skeletal myocytes are Not detected (HPA: tissue IHC). Compare the named cell populations, since a whole tissue contains more than one cell type. |
| How strong is the tissue-pattern evidence? | HPA labels its tissue IHC profile Approved, while noting low staining–RNA consistency and pending external verification (HPA: tissue reliability). Use the pattern as an expectation, not a specimen-level guarantee. |
| What can the antibody record support? | HPA042303 is listed as IHC Approved and ICC Supported (HPA: antibodies). The supplied record provides no IHC Enhanced designation or second-antibody confirmation; unexpected patterns merit an independent specificity check. |
| What does topology imply for reading the stain? | ACAA2 has no annotated transmembrane segment and is assigned to mitochondria (UniProt P42765 topology/location). A dominant membrane rim is therefore unexpected; topology does not establish which retrieval condition works. |
| IF/ICC: where should signal localize? | The main location is mitochondria; cytosol and basal body are additional, uncertain assignments (HPA: subcellular ICC-IF). This is an interpretation cue for the separate IF/ICC guide, not an IHC protocol option. |
| Is ACAA2-specific fixation sensitivity known? | No target-specific fixation effect is supplied by UniProt P42765 or HPA tissue IHC. General IHC practice may assess retrieval and processing when a run fails, without attributing that failure to a proven ACAA2 fixation effect. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive cells are unstained. | A failed detection run or unsuitable staining conditions are possibilities; HPA reports High staining in hepatocytes and kidney tubule cells (HPA: tissue IHC). | Check an on-run positive tissue control and detection reagents. If the run failed, review the catalog antibody’s IHC-P instructions and adjust one general IHC variable at a time. |
| The whole section looks uniformly brown. | General IHC practice: excessive detection signal, residual endogenous activity, or incomplete washing can obscure a cellular pattern. | Compare with a no-primary control, inspect the counterstain and chromogen development, and review blocking and washes. Look again for the granular cytoplasmic pattern reported by HPA (HPA: tissue IHC). |
| Strong colour appears in HPA not-detected cells. | Possible cross-reactivity or endogenous detection activity; adipocytes and skeletal myocytes are Not detected in the reported tissue profile (HPA: tissue IHC). | Identify the stained cell type on the section, compare a no-primary control, and seek an independent specificity check before scoring that signal as ACAA2. |
| Signal is mainly nuclear or outlines cell membranes. | The dominant compartment conflicts with mitochondrial localization (UniProt P42765; HPA: subcellular ICC-IF). | Review staining controls and morphology, then check whether expected positive cells show cytoplasmic granules. Do not score the discordant compartment as a routine positive pattern. |
| Signal is weak in a named positive cell population. | A weak run or biological variation may be involved; HPA’s High category describes its observations, not a guaranteed intensity in every specimen (HPA: tissue IHC). | Compare the same run’s positive control, cell morphology, and background. Review the catalog antibody’s IHC-P conditions before changing interpretation thresholds. |
| Positive control stains, but the test tissue does not. | The tested cell population may differ from HPA’s named positive populations, or the specimen may truly show less detectable signal (HPA: tissue IHC). | Score the relevant cells rather than the tissue as a whole, record the compartment and intensity, and report the result with HPA’s pending external-verification caveat (HPA: tissue reliability). |
Experiment evidence
IHC & IF images showing ACAA2 staining
M08341-1 · IHCIHC analysis of ACAA2 using anti-ACAA2 antibody (M08341-1). ACAA2 was detected in a paraffin-embedded section of human colorectal adenocarci
M08341-1 · IHCIHC analysis of ACAA2 using anti-ACAA2 antibody (M08341-1). ACAA2 was detected in a paraffin-embedded section of human colung cancer tissue.
M08341-1 · IFIF analysis of ACAA2 using anti-ACAA2 antibody (M08341-1). ACAA2 was detected in an immunocytochemical section of Hela cells. Enzyme antigen
PB10022 · IHCIHC analysis of ACAA2 using anti-ACAA2 antibody (PB10022). ACAA2 was detected in paraffin-embedded section of human intestinal cancer tissue
PB10022 · IFIF analysis of ACAA2 using anti-ACAA2 antibody (PB10022). ACAA2 was detected in immunocytochemical section of U20S cell. Enzyme antigen retr
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000167315-ACAA2/tissue/cerebral+cortex#imid_11124423
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000167315-ACAA2/tissue/colon#imid_11122397
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000167315-ACAA2/tissue/liver#imid_11122398
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000167315-ACAA2/tissue/kidney#imid_11122415
HPA · ICC-IF CACO-2Human Protein Atlas immunofluorescence, CACO-2, antibody HPA042303; green = ACAA2, blue = nucleus, red = microtubules.
HPA · ICC-IF CACO-2Human Protein Atlas immunofluorescence, CACO-2, antibody HPA042303; green = ACAA2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ACAA2 IHC & IF
🧪Run kidney first: cells in tubules should stain strongly (HPA: High in kidney cells in tubules). Use lymph node germinal center cells as the negative tissue comparator (HPA: Not detected in lymph node germinal center cells); on the kidney slide, non-tubular cells should show only background staining to serve as internal negatives, but HPA does not designate those cells ACAA2-negative (HPA: kidney call applies to cells in tubules).
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 ACAA2 in CACO-2, Hep-G2, U2OS, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), NIH 3T3, with annotated localisation: Mitochondria (supported) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, a concentration-matched rabbit IgG isotype control, and ACAA2 knockout material as a biological negative (caption: rabbit anti-ACAA2 M08341-1; standard IHC specificity controls). Quench endogenous peroxidase and check for residual background in the kidney section (caption: HRP/DAB detection; standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected M08341-1 tissue-IHC caption does not state a fixative (caption: fixative not stated). The paraffin-section example used heat retrieval in EDTA at pH 8.0 and a 1:50 primary dilution overnight at 4°C; it does not establish that retrieval is mandatory (caption: M08341-1 tissue IHC). The supplied evidence does not establish frozen sections or IF/ICC as easier; ICC-IF supports mitochondrial localization, while kidney HRP/DAB staining requires a check for endogenous peroxidase background (HPA: mitochondria supported; caption: HRP/DAB; standard chromogenic IHC practice).
HPA tissue IHC evidence for ACAA2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low 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 → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Kidney | Cells in tubules | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ACAA2 IHC Tips
Troubleshoot ACAA2 staining in paraffin sections by checking retrieval, controls, compartment, and scoring before interpreting chromogenic signal.
Which retrieval condition should I start with for ACAA2 paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet M08341-1). The selected image used this condition before overnight incubation at 4°C with the catalog antibody at 1:50 (datasheet M08341-1). If staining is weak, compare retrieval duration on adjacent sections while holding antibody concentration, detection, and development constant (standard IHC practice). Check tissue integrity alongside signal: excessive heating can damage section morphology and make granular cytoplasmic staining difficult to judge (standard IHC practice; HPA: granular cytoplasmic profile). Record retrieval time and heating method for every slide so a change in signal can be traced to the retrieval step (standard IHC practice).
How should I assess whether fixation is affecting ACAA2 staining?
The selected tissue caption reports a paraffin-embedded section but does not state its fixative, so ACAA2 sensitivity to fixation remains unknown (datasheet M08341-1). Record the fixative, fixation duration, tissue thickness, and processing history for each case before comparing staining intensity (standard IHC practice). When possible, compare sections processed together and include the same positive reference tissue in each run to detect a processing-related loss of signal (standard IHC practice; HPA: high staining in liver hepatocytes). Do not attribute weak staining to a specific fixation effect based on the reported mitochondrial location or modified residues; those annotations establish neither fixation sensitivity nor an optimal fixation time (UniProt P42765: mitochondrion, modified residues).
What staining pattern is plausible for ACAA2, and what pattern needs investigation?
Look for granular cytoplasmic staining in the relevant cells, consistent with the tissue IHC profile and supported mitochondrial localisation (HPA: granular cytoplasmic profile; HPA subcellular: mitochondria supported). ACAA2 is annotated as mitochondrial and lacks a transmembrane segment, but the exact epitope of the catalog antibody is not supplied (UniProt P42765 topology; datasheet M08341-1). Compare the signal with a mitochondrial marker on a matched section when compartment assignment is uncertain (standard IHC practice; UniProt P42765: mitochondrion). Predominantly nuclear staining or a smooth, uniform cytoplasmic deposit warrants review of antibody concentration, chromogen development, and tissue background before it is called ACAA2 (standard IHC practice; HPA: granular cytoplasmic profile).
Could isoforms or epitope accessibility explain inconsistent ACAA2 staining?
The record lists 0 isoforms and one annotated chain spanning residues 1–397, so isoform-specific staining cannot be inferred from these data (UniProt P42765: isoforms and processing). ACAA2 has no annotated transmembrane segment, while its exact antibody epitope is unspecified; that limits predictions about retrieval-dependent epitope exposure (UniProt P42765 topology; datasheet M08341-1). Acetylation, succinylation, and phosphorylation are annotated, but no supplied evidence shows that any modification changes this antibody’s IHC binding (UniProt P42765: modified residues; datasheet M08341-1). If samples stain inconsistently, first compare retrieval, processing, and run controls, then seek epitope information before proposing a modification-specific explanation (standard IHC practice).
How can IF help check an ambiguous chromogenic ACAA2 result?
Use a separate IF experiment to compare ACAA2 with a mitochondrial marker and a marker identifying the cell population being assessed in the paraffin section (HPA subcellular: mitochondria supported; standard IF practice). Select spectrally separated fluorophores and inspect an unstained section for tissue autofluorescence before assigning a weak granular signal (standard IF practice). Permeabilisation should permit antibody access to an intracellular mitochondrial epitope, but its precise membrane-facing position is unavailable because the antibody epitope is unspecified (UniProt P42765: mitochondrion; datasheet M08341-1). Treat IF colocalisation as supporting evidence for the chromogenic interpretation, and retain single-channel controls when judging bleed-through or background (standard IF practice).
How do I troubleshoot diffuse or granular background in ACAA2 IHC?
First compare the stained section with a no-primary control to identify signal arising from detection reagents or endogenous tissue activity (standard IHC practice). The selected paraffin-section example used 10% goat serum blocking, peroxidase-linked secondary detection, and DAB; these are reported workflow conditions, not proof that every specimen will have low background (datasheet M08341-1). Include a peroxidase-blocking step and assess the no-primary control before lengthening DAB development, since endogenous peroxidase can produce chromogenic signal (standard IHC practice). If background persists, optimise washing and primary-antibody concentration against a positive reference while checking that true granular cytoplasmic staining remains visible (standard IHC practice; HPA: granular cytoplasmic profile).
How should I quantify ACAA2 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and analysis region before scoring, then apply the same thresholds and image settings to every section (standard IHC practice). Report the percentage of positive cells and an H-score from 0–300, or measure positive-cell density per mm² when cell counts are the relevant endpoint (standard IHC practice). Normalise positive counts to the number of eligible cells, or to analysed tissue area for density, and exclude folds, necrosis, and section edges consistently (standard IHC practice). Record granular cytoplasmic signal separately from nonspecific deposits, and stratify by cell type because HPA reports high staining in liver hepatocytes and kidney tubular cells (HPA: liver and kidney tissue IHC; HPA: granular cytoplasmic profile).
When is ACAA2 staining convincing rather than an artefact?
A convincing result shows reproducible granular cytoplasmic staining in morphologically intact cells, with low signal in the no-primary control (HPA: granular cytoplasmic profile; standard IHC practice). Supported mitochondrial localisation makes predominantly nuclear staining a reason to investigate specificity, while HPA’s additional cytosol and basal-body assignments are marked uncertain (HPA subcellular: mitochondria supported; cytosol and basal body uncertain). Examine section edges and necrotic areas for deposits, and check the peroxidase-blocking control when DAB signal could reflect endogenous enzyme activity (standard IHC practice). Use tissue comparisons cautiously: HPA lists high hepatocyte staining but also flags low consistency between antibody staining and RNA expression, with external verification pending (HPA: liver hepatocytes high; reliability description).
Boster reagents
Best ACAA2 / 3-ketoacyl-CoA thiolase, mitochondrial IHC Antibodies
Both catalog antibodies have IHC figures from human paraffin sections and IF/ICC figures from human cell samples; both list Human, Mouse and Rat reactivity (catalog image captions; catalog reactivity).
Real IHC data
Anti-ACAA2 Rabbit Monoclonal Antibody
Cat # M08341-1
Real IHC data
Anti-ACAA2 Antibody ®
Cat # PB10022
M08341-1 will render with IHC data from a human colorectal adenocarcinoma paraffin section; its IF/ICC figure uses HeLa cells (M08341-1 image captions). PB10022 will render with IHC data from human intestinal cancer paraffin sections; its IF/ICC figure uses U20S cells (PB10022 image captions).
Which to pick: For tissue IHC, choose M08341-1 for the documented 1:50 EDTA pH 8 workflow, or PB10022 for the documented 1 μg/ml citrate pH 6 workflow; both captions show paraffin sections and leave the fixative unreported (respective IHC image captions). For IF/ICC, M08341-1 is a rabbit monoclonal with a HeLa cell figure, while PB10022 has a U20S cell figure and no clone specified (catalog titles and IF image captions). Both list Human, Mouse and Rat reactivity, but their supplied IHC and IF figures show human samples only (catalog reactivity and image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P42765 (THIM_HUMAN, 3-ketoacyl-CoA thiolase, mitochondrial).
- Human Protein Atlas. ACAA2 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ACAA2 subcellular location (ICC-IF): Mainly localized to the mitochondria. In addition localized to the cytosol and basal body..
- Human Protein Atlas. ACAA2 antibody validation summary (1 antibodies).
- The metabolic crossroads: glycolysis in cancer and neurodegeneration. BMC neurology 2025 — PMC12771824.
- Acetyl-CoA acyltransferase 2 palmitoylation drives liver fibrosis by inducing hepatic stellate cell ferroptosis. Redox biology 2026 — PMC12859212.
- Metabolic classifications of renal cell carcinoma reveal intrinsic connections with clinical and immune characteristics. Journal of translational medicine 2023 — PMC9960222.
- ACAA2 Protects Against Cardiac Dysfunction and Lipid Peroxidation in Renal Insufficiency with the Treatment of S-Nitroso-L-Cysteine. Biomolecules 2025 — PMC11940541.
- PubMed PMID:8241273 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:15242332 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ACAA2 (3-ketoacyl-CoA thiolase, mitochondrial). Expected localisation Granular cytoplasm observed (HPA tissue IHC); mitochondrion annotated (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M08341-1). Validated IHC antibody: M08341-1 at 1:50 (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 colorectal adenocarcinoma tissue; fixative not specified (datasheet M08341-1); 2. Fixation: Image fixative and duration unreported (datasheet M08341-1); 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: EDTA pH 8.0 (datasheet M08341-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M08341-1); 8. Primary antibody: Rabbit monoclonal (clone 26A90) anti-ACAA2, 1:50 (datasheet M08341-1); 9. Primary incubation: Overnight at 4 °C (datasheet M08341-1). Watch for: Fixation: Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific); Caveat: Antibody staining has low concordance with RNA (HPA tissue IHC); Isoform / epitope: No annotated isoforms; no isoform-dependent epitope map (UniProt); Regulation: No expression regulator annotated (UniProt). Evidence: UniProt P42765, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the mitochondria. In addition localized to the…, Datasheet M08341-1, PMC12771824, PMC11940541. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ACAA2/immunohistochemistry-ihc-guide.