ACAT1 / Acetyl-CoA acetyltransferase, mitochondrial · IHC design guide

Design Immunohistochemistry for ACAT1

Plan ACAT1 chromogenic IHC in paraffin sections using the observed granular cytoplasmic tissue pattern (HPA tissue IHC). Compare staining across tissues with kidney and liver RNA enrichment in mind (HPA tissue IHC).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for ACAT1 (IHC for ACAT1): expected localisation Granular cytoplasm (HPA tissue IHC); mitochondria (UniProt), antibody A02008-1, validated IHC image, and IHC protocol steps — Printable **ACAT1** IHC protocol sheet — expected localisation Granular cytoplasm (HPA tissue IHC); mitochondria (UniProt), antibody A02008-1, controls and protocol steps. Open the full ACAT1 IHC guide →

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ACAT1 Immunohistochemistry Experimental Design Guide

Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.

Must know before staining

	Expected localisation	Granular cytoplasm (HPA tissue IHC); mitochondria (UniProt)
	Staining pattern	Ubiquitous granular cytoplasmic staining (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A02008-1)
	Positive control ⓘ	Adrenal gland+4 more · see all
	Negative control ⓘ	None in HPA (detected in all 45 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	Smooth muscle cells show low staining (HPA tissue IHC)
	Regulation	Kidney and liver RNA enhanced (HPA tissue RNA)
	Isoform / epitope	2 isoforms; mature chain starts at residue 34; epitope coverage unknown (UniProt)

Section 1

Recommended ACAT1 IHC & IF Protocols

The catalog antibody’s IHC protocol is accompanied by four published staining examples. Check antibody identity when adapting phospho-ACAT1 or SOAT1/ACAT1 methods (PMC11544177; PMC8994887).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet A02008-1)
Fixation	Image fixative and duration unreported (datasheet A02008-1); 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 A02008-1); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A02008-1)
Primary antibody	Rabbit anti-ACAT1, 2-5 μg/ml (datasheet A02008-1)
Primary incubation	Overnight at 4 °C (datasheet A02008-1)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet A02008-1)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	ACAT1-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression with a granular pattern. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet: A02008-1); the published citrate methods are alternatives to evaluate (PMC8994887; PMC6795108; PMC13614741).

Section 2

What Is the Expected ACAT1 Staining Pattern?

ACAT1 is mitochondrial and has no transmembrane segment (UniProt P24752 topology). In paraffin-section IHC, expect granular cytoplasmic staining across many cell types (HPA: ubiquitous cytoplasmic expression with a granular pattern). Documented high-staining examples include adrenal glandular cells, bronchial respiratory epithelial cells, and cerebellar Purkinje cells (HPA: High in each). HPA rates the tissue staining evidence Enhanced, based on consistency with RNA expression (HPA: Enhanced reliability).

What am I looking at on my slide?
Granular cytoplasmic stain in adrenal glandular or bronchial respiratory epithelial cells.	This matches documented High IHC staining in those cells (HPA: tissue IHC) and the expected mitochondrial distribution (UniProt P24752: mitochondrion). Judge the granules against nearby unstained structures and the negative control (general IHC practice).
Predominantly nuclear or sharply cell-surface staining, without cytoplasmic granules.	That compartment conflicts with the mitochondrial assignment (UniProt P24752) and HPA's granular cytoplasmic profile (HPA: tissue IHC). Check morphology and the antibody's staining controls before interpreting it as ACAT1 (general IHC practice).
Strong stain concentrated in smooth muscle cells.	HPA reports Low staining in smooth muscle cells (HPA: tissue IHC). Unexpected strong staining may reflect cross-reactivity or detection background; compare it with documented high-staining cells on the same run (general IHC practice).
Uniform cytoplasmic haze obscures the granular pattern.	The haze prevents a confident match to HPA's granular profile (HPA: tissue IHC). Excess antibody or detection background are possible general IHC causes; compare a titration and negative control (general IHC practice).
No stain in a documented high-staining tissue.	An absent signal in adrenal glandular, bronchial respiratory epithelial, or cerebellar Purkinje cells warrants a run check (HPA: High in each). Review section integrity, retrieval, antibody dilution, and detection controls (general IHC practice).

💡Expected ACAT1 appearanceCall a positive result when cytoplasmic granules appear in documented high-staining cells at clear intensity (HPA: tissue IHC); dominant nuclear or cell-surface stain is discordant with mitochondrial ACAT1 (UniProt P24752; HPA: subcellular).

How each factor affects the staining
Which compartment should guide IHC scoring?	Score granular cytoplasmic signal (HPA: tissue IHC). Mitochondrial localization supports that pattern (UniProt P24752); a chromogenic section need not resolve individual mitochondria (general IHC practice).
Which tissues offer a documented comparison?	HPA records High stain in adrenal glandular, bronchial respiratory epithelial, and cerebellar Purkinje cells, among others; smooth muscle cells are Low (HPA: tissue IHC). These are comparisons, not absolute positive or negative controls.
How strong is the antibody-level IHC evidence?	HPA004428 and HPA007569 each carry Enhanced IHC validation (HPA: antibody records). That supports the reported pattern, but the payload gives no validation status for a different catalog antibody.
Do processing or isoforms change the predicted slide pattern?	UniProt lists a mature chain at residues 34–427 and 2 isoforms (UniProt P24752). The payload gives no epitope map or isoform-specific staining evidence, so their effects on this IHC result are unknown.
IF/ICC: what localization should be seen?	Mitochondrial staining is expected (HPA: subcellular ICC-IF, enhanced). HPA lists images for A-431, U-251MG, U2OS, and KOLF2.1J; that IF observation supports a compartment comparison, not an IHC protocol.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Documented high-staining cells show no chromogenic signal.	A failed detection step, unsuitable dilution, or ineffective retrieval is possible (general IHC practice); HPA reports these cells as High (HPA: tissue IHC).	Confirm that a same-run positive section stains, then check reagent sequence and optimize dilution or retrieval using the antibody's IHC-P instructions (general IHC practice).
Signal is mainly nuclear or outlines cell borders.	The location disagrees with mitochondrial ACAT1 (UniProt P24752) and HPA's granular cytoplasmic profile (HPA: tissue IHC).	Recheck cell boundaries and compare with a negative control; reassess antibody concentration and specificity if the discordant pattern persists (general IHC practice).
Smooth muscle stains as strongly as the intended positive cells.	HPA records smooth muscle cells as Low (HPA: tissue IHC); strong signal could include nonspecific binding or detection background (general IHC practice).	Compare documented high-staining cells and negative controls on the same run, then titrate the primary antibody (HPA: tissue IHC; general IHC practice).
Brown haze fills cells or the surrounding section.	Background may obscure the documented granular cytoplasmic pattern (HPA: tissue IHC); excess primary antibody or detection reagent is possible (general IHC practice).	Compare a no-primary control, review blocking and washes, and titrate the primary antibody or detection development (general IHC practice).
Brown deposits remain in the no-primary control.	Primary-independent signal can arise from endogenous detection activity or deposited reagent (general IHC practice).	Check the detection system's endogenous-activity block, reagent handling, and no-primary control before scoring ACAT1 (general IHC practice).
The section stains broadly, with no obvious negative tissue.	Broad staining can fit HPA's ubiquitous cytoplasmic profile; the supplied HPA record lists no negative tissue (HPA: tissue IHC).	Score compartment and granularity, then compare documented High cells with Low smooth muscle cells and run controls (HPA: tissue IHC; general IHC practice).

Source: UniProt P24752 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing ACAT1 staining

IHC analysis of ACAT1 using anti-ACAT1 antibody (A02008-1). ACAT1 was detected in a paraffin-embedded section of human liver cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-ACAT1 Antibody (A02008-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

A02008-1 · IHCIHC analysis of ACAT1 using anti-ACAT1 antibody (A02008-1). ACAT1 was detected in a paraffin-embedded section of human liver cancer tissue.

IHC analysis of ACAT1 using anti-ACAT1 antibody (A02008-1). ACAT1 was detected in a paraffin-embedded section of human non-small cell lung cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-ACAT1 Antibody (A02008-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

A02008-1 · IHCIHC analysis of ACAT1 using anti-ACAT1 antibody (A02008-1). ACAT1 was detected in a paraffin-embedded section of human non-small cell lung c

IF analysis of ACAT1 using anti-ACAT1 antibody (A02008-1). ACAT1 was detected in an immunocytochemical section of HELA cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-ACAT1 Antibody (A02008-1) overnight at 4°C. Cy3 Conjugated Goat Anti-Rabbit IgG (BA1032) was used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

A02008-1 · IFIF analysis of ACAT1 using anti-ACAT1 antibody (A02008-1). ACAT1 was detected in an immunocytochemical section of HELA cells. Enzyme antigen

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000075239-ACAT1/tissue/cerebral+cortex#imid_1612128

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000075239-ACAT1/tissue/colon#imid_1612054

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000075239-ACAT1/tissue/liver#imid_1612063

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000075239-ACAT1/tissue/kidney#imid_1612064

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA004428; green = ACAT1, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for ACAT1 IHC & IF

🧪Run bronchus first; respiratory epithelial cells should stain (HPA: High in bronchus respiratory epithelial cells). HPA detects ACAT1 in all 45 scored tissues, so there is no supported negative tissue (HPA: no negative rows); no internal negative cell type is established, but cells without specific signal should retain counterstain without granular cytoplasmic DAB staining (UniProt P24752: mitochondrion; standard IHC practice).

Positive control tissue: Adrenal gland (Glandular cells, HPA High)

Negative control tissue: None in HPA: ACAT1 is detected in all 45 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 ACAT1 in A-431, U-251MG, U2OS, KOLF2.1J, with annotated localisation: Mitochondria (enhanced) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control and a rabbit IgG isotype control matched to the primary antibody’s clonality and concentration (caption: rabbit anti-ACAT1; standard IHC practice). Use ACAT1-knockout material if available, or validated peptide competition, as a biological specificity control; quench endogenous peroxidase and check bronchial mucus for background with HRP/DAB detection (caption: HRP/DAB; standard IHC practice).

⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected paraffin-section caption does not state a fixative (caption: fixative not stated). The reported IHC procedure uses heat retrieval in EDTA at pH 8.0 (caption: EDTA pH 8.0); HPA ICC-IF images support mitochondrial localization (HPA: Mitochondria), but the supplied evidence does not establish that frozen sections or IF are easier. In bronchus, inspect luminal mucus for nonspecific DAB deposition (standard IHC practice).

HPA tissue IHC evidence for ACAT1

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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 →
Appendix	Glandular cells	High	Protein (IHC)	HPA →
Bronchus	Respiratory epithelial cells	High	Protein (IHC)	HPA →
Caudate	Neuronal cells	High	Protein (IHC)	HPA →
Cerebellum	Purkinje cells	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
None in HPA: ACAT1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

Section 3

Advanced ACAT1 IHC Tips

Troubleshoot chromogenic ACAT1 staining in paraffin sections using the catalog antibody’s tissue IHC conditions and the expected granular mitochondrial pattern (datasheet A02008-1; HPA tissue IHC; HPA subcellular).

How should I retrieve ACAT1 when staining is weak in paraffin sections?

Start with heat-mediated retrieval in EDTA at pH 8.0 (datasheet A02008-1). The selected tissue IHC image used this retrieval before an overnight incubation at 4°C with 2 μg/ml primary antibody, providing a documented starting point for the catalog antibody (datasheet A02008-1). If staining remains weak, first check retrieval temperature, heating duration, cooling, and section adherence while keeping buffer and pH fixed (standard IHC practice). Only then compare an alternative retrieval condition on adjacent sections, judging both granular cytoplasmic signal and tissue damage against the documented condition (HPA tissue IHC; standard IHC practice).

Could fixation be masking ACAT1 in my paraffin sections?

Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative, and the supplied evidence does not establish one (datasheet A02008-1). Record the actual fixation method and duration for each specimen before comparing staining across blocks (standard IHC practice). If similarly processed sections stain differently, assess tissue preservation and repeat the documented EDTA pH 8.0 retrieval under consistent heating conditions before attributing the difference to fixation (datasheet A02008-1; standard IHC practice). Compare adjacent sections with matched detection and examine whether the expected granular cytoplasmic pattern survives, without treating that pattern as evidence of fixation tolerance (HPA tissue IHC; standard IHC practice).

What staining pattern should I accept as ACAT1 positive?

Expect granular cytoplasmic staining in chromogenic tissue IHC, consistent with ACAT1’s mitochondrial localisation (HPA tissue IHC; UniProt P24752 subcellular location). The protein has no annotated transmembrane segment, so a crisp cell-surface outline alone is a poor match for its documented location (UniProt P24752 topology; standard IHC interpretation). Assess granules within intact cell bodies at high magnification and compare their distribution with the same section’s morphology and counterstain (HPA tissue IHC; standard IHC practice). High staining is reported in several cell populations, including adrenal glandular cells and cerebellar Purkinje cells; use the relevant tissue context instead of requiring identical intensity in every cell (HPA tissue IHC).

Can this staining distinguish ACAT1 isoforms or a modified epitope?

Do not assign an isoform from this chromogenic pattern alone: ACAT1 has 2 annotated isoforms, while the supplied tissue IHC caption does not specify the antibody epitope or demonstrate isoform discrimination (UniProt P24752 isoforms; datasheet A02008-1). The annotated processed chain spans residues 34–427, and multiple modified residues are reported, including alternative modifications at residues 66 and 174 (UniProt P24752 processing and modified residues). Those annotations identify questions for epitope review; they do not establish that a modification changes staining (UniProt P24752; standard IHC interpretation). For an isoform-specific claim, obtain epitope mapping and independently validated isoform-specific evidence before interpreting section scores (standard IHC validation practice).

How can I adapt this ACAT1 question to multiplex immunofluorescence?

Treat the catalog antibody’s IF performance as unestablished by the supplied IHC caption; the separate subcellular evidence places ACAT1 in mitochondria (datasheet A02008-1; HPA subcellular). Multiplex an ACAT1 channel with a marker for the expected cell population, such as a glandular-cell marker in a tissue where glandular staining is reported, and check both channels separately (HPA tissue IHC; standard IF practice). Choose a spectrally separated, preferably far-red fluorophore when tissue autofluorescence obscures shorter-wavelength signal, with single-channel controls (standard IF practice). Because the epitope’s membrane-facing side is unspecified, optimise mild permeabilisation and verify access to the mitochondrial epitope without assuming its precise intramitochondrial location (UniProt P24752 topology; standard IF practice).

How do I reduce diffuse brown background without losing ACAT1?

The selected image used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, a peroxidase-conjugated secondary for 30 minutes at 37°C, and DAB detection (datasheet A02008-1). Reproduce those documented conditions first, then titrate the primary or adjust washes on adjacent sections if diffuse staining persists (datasheet A02008-1; standard IHC practice). Include a no-primary control and block endogenous peroxidase before HRP detection to distinguish reagent background from tissue enzyme activity (standard IHC practice). Judge improvement by preservation of granular cytoplasmic staining in intact cells, rather than by a uniformly pale section (HPA tissue IHC; standard IHC interpretation).

What should I score when ACAT1 staining varies across a section? ⚠ ANSWER MARKED FOR VERIFICATION

Define the scored cell population and viable tissue area before measuring chromogenic signal, then apply the same rules to every section (standard IHC quantification practice). An H-score combines the percentage of cells at intensities 0–3 into a 0–300 score; alternatively, report percent positive cells or positive-cell density per mm² of viable tissue (standard IHC quantification practice). Score granular cytoplasmic staining within cells, and record the fraction excluded for folds, edges, or necrosis (HPA tissue IHC; standard IHC quantification practice). Normalise counts to the defined cell population or viable area, and compare sections only under matched staining and imaging conditions (standard IHC quantification practice).

How can I separate genuine ACAT1 staining from artefact?

A plausible ACAT1 signal is granular and cytoplasmic in intact cells, matching the reported tissue pattern and mitochondrial localisation (HPA tissue IHC; HPA subcellular). Check whether the stained cells fit the tissue context: high signal is documented in several glandular populations, whereas smooth muscle cells are listed as low (HPA tissue IHC). Treat staining confined to section edges, necrotic regions, or a sharp cell-surface rim as suspect until it reproduces in well-preserved interior tissue (UniProt P24752 topology; standard IHC interpretation). If brown signal persists without primary antibody, investigate endogenous peroxidase or detection background before calling those cells ACAT1 positive (standard IHC practice).

Boster reagents

Best ACAT1 / Acetyl-CoA acetyltransferase, mitochondrial IHC Antibodies

A02008-1 has IHC images from human paraffin sections and IF/ICC data from HELA cells (catalog captions); its listed reactivity covers human, mouse and rat (catalog).

Real IHC data

Anti-ACAT1 Antibody ®

Cat # A02008-1

View product →

The A02008-1 card shows IHC in human liver cancer tissue; additional captions document human non-small cell lung cancer and placenta paraffin sections (A02008-1 IHC captions). Its IF/ICC caption documents HELA cells (A02008-1 IF caption).

Which to pick: For tissue IHC, choose A02008-1: its own paraffin-section captions document EDTA pH 8 retrieval and 2 μg/ml primary antibody; the fixative is unreported (A02008-1 IHC captions). For IF/ICC, choose A02008-1 at the listed 5 μg/ml; M02008 lists IHC but no IF/ICC application or image (catalog). For mouse or rat samples, both SKUs list reactivity, but the supplied IHC images document human tissue only; M02008 is monoclonal, while A02008-1 has no clone listed (catalog).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry P24752 (THIL_HUMAN, Acetyl-CoA acetyltransferase, mitochondrial).
Human Protein Atlas. ACAT1 tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. ACAT1 subcellular location (ICC-IF): Localized to the mitochondria..
Human Protein Atlas. ACAT1 antibody validation summary (2 antibodies).
Target fishing and mechanistic insights of the natural anticancer drug candidate chlorogenic acid. Acta pharmaceutica Sinica. B 2024 — PMC11544177.
Assessment of the diagnostic and prognostic relevance of ACAT1 and CE levels in plasma, peritoneal fluid and tumor tissue of epithelial ovarian cancer patients - a pilot study. BMC cancer 2022 — PMC8994887.
ACAT1 and Metabolism-Related Pathways Are Essential for the Progression of Clear Cell Renal Cell Carcinoma (ccRCC), as Determined by Co-expression Network Analysis. Frontiers in oncology 2019 — PMC6795108.
Cholesterol metabolism dysregulation in eyelid sebaceous gland carcinoma and its clinical significance. Indian journal of ophthalmology 2026 — PMC13614741.
PubMed PMID:1979337 — UniProt-cited evidence.
PubMed PMID:1684944 — UniProt-cited evidence.
PubMed PMID:14702039 — UniProt-cited evidence.