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ACLY / ATP-citrate synthase · IHC design guide
Design Immunohistochemistry for ACLY
Plan chromogenic ACLY IHC in paraffin sections around the cytoplasmic and nuclear staining seen across tissues (HPA tissue IHC). Pancreatic endocrine cells provide a high staining reference (HPA tissue IHC); the catalog antibody PB10024 has an IHC working range of 1–2 µg/mL (datasheet: PB10024).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ACLY 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 and nuclear tissue staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic/nuclear signal; pancreatic endocrine cells stain high (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PB10024) | |
| Positive control | Pancreas+4 more · see all | |
| Negative control | Esophagus+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific). Selected-image fixative and duration unreported (datasheet PB10024); verify before use. | |
| Caveat | Antibody staining has low concordance with RNA expression (HPA tissue IHC) | |
| Regulation | No expression regulator annotated (UniProt) | |
| Isoform / epitope | 3 isoforms; epitope coverage is unresolved (UniProt) |
Section 1
Recommended ACLY IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: PB10024) is accompanied by published ACLY staining protocols for lung cancer (PMC4427316) and mouse xenograft sections (PMC6774899).
| Sample | Paraffin-embedded human colonic adenocarcinoma tissue; fixative not specified (datasheet PB10024) |
| Fixation | Image fixative and duration unreported (datasheet PB10024); 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 PB10024); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB10024) |
| Primary antibody | Rabbit anti-ACLY, 1-2μg/ml (datasheet PB10024) |
| Primary incubation | Overnight at 4 °C (datasheet PB10024) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet PB10024) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ACLY-positive staining in pancreatic endocrine cells of pancreas (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and nuclear expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval (datasheet: PB10024); the published protocols used pH 9.0 (PMC4427316) and pH 6.0 (PMC6774899).
Section 2
What Is the Expected ACLY Staining Pattern?
ACLY is a cytosolic enzyme with no transmembrane segment (UniProt P53396: cytosol; topology). In paraffin-section IHC, expect predominantly cytoplasmic staining, with nuclear staining possible in several tissues (HPA: tissue IHC). Pancreatic endocrine cells, prostate glandular cells and testicular Leydig cells are reported as High (HPA: tissue IHC). Interpret tissue comparisons cautiously: HPA rates IHC reliability Enhanced but reports low consistency between staining and RNA expression (HPA: reliability description).
| Clear cytoplasmic signal in pancreatic endocrine cells, prostate glandular cells or Leydig cells. | This matches the reported High-staining cell populations (HPA: tissue IHC) and ACLY's cytosolic location (UniProt P53396: subcellular location). Compare signal with adjacent tissue and the negative control under the same detection conditions (general IHC practice). |
| Predominantly membrane-confined staining, with little cytoplasmic signal, in a reported positive population. | A membrane-only pattern conflicts with cytosolic localization and the absence of a transmembrane segment (UniProt P53396: subcellular location; topology). Review morphology and controls before calling it ACLY. Nuclear signal alone is less decisive: HPA also reports nuclear tissue staining (HPA: tissue IHC). |
| Strong staining in a cell population reported as Not detected, such as esophageal squamous epithelium. | This is discordant with that HPA observation (HPA: esophagus, squamous epithelial cells, Not detected). Cross-reactivity or endogenous chromogen-producing activity are possibilities, not diagnoses (general IHC practice). Check the no-primary control and assess whether the expected positive cell population also stains. |
| Diffuse color across cells and extracellular areas, obscuring cell boundaries. | This is background rather than an interpretable cell-specific pattern (general IHC practice). Examine the no-primary control for detection-system signal, then review blocking, washes and chromogen development (general IHC practice). HPA tissue levels do not identify the cause of background (HPA: tissue IHC). |
| No visible signal in a pancreatic endocrine-cell positive control. | That result disagrees with the reported High staining in those cells (HPA: pancreas). Before scoring the study samples negative, verify that the target cell population is present and assess retrieval, primary-antibody conditions and detection performance using the same run (general IHC practice). |
💡Expected ACLY appearanceCall a result positive when cytoplasmic staining is discernible in a reported High population, such as pancreatic endocrine cells, with nuclear staining permissible in tissue IHC (HPA: pancreas; tissue IHC); a membrane-only pattern or equal color throughout cell-free areas calls for control review (UniProt P53396: topology; general IHC practice).
| Compartment and topology (UniProt P53396: subcellular location; topology). | Cytosolic localization and no transmembrane segment favor a cytoplasmic readout (UniProt P53396). HPA additionally observes nuclear staining in several tissues (HPA: tissue IHC); do not reject every nucleus-positive section solely on compartment. |
| Choice of tissue and cell population (HPA: tissue IHC). | Pancreatic endocrine cells, prostate glandular cells and Leydig cells are reported High; kidney tubule cells and small-intestinal glandular cells are Medium (HPA: tissue IHC). Esophageal squamous cells and rectal glandular cells are reported Not detected (HPA: tissue IHC). Score the specified cells, rather than an entire organ. |
| Strength of the tissue evidence (HPA: reliability description; antibody validation). | HPA labels the tissue IHC profile Enhanced while noting low consistency between antibody staining and RNA expression (HPA: reliability description). Four listed antibodies have Enhanced IHC validation; CAB007783 is Approved (HPA: antibodies). These labels support use of the pattern as a comparator, not a guarantee for every specimen. |
| IF/ICC expectation? (HPA: subcellular ICC-IF). | HPA reports cytosol as the supported main location in ICC-IF; nucleoplasm is uncertain, and the principal piece is an approved additional location (HPA: subcellular ICC-IF). Use that information to interpret an IF image; this section supplies no IF/ICC protocol. |
| Sequence variation and modification (UniProt P53396: isoforms; modified residues). | UniProt lists three isoforms and modified residues, including phosphorylation sites (UniProt P53396). The supplied record gives no epitope or staining effect for a particular IHC antibody, so these annotations cannot explain a changed section pattern without separate evidence. |
| Situation | Likely cause | Next action |
|---|---|---|
| The positive-control section has no chromogen in pancreatic endocrine cells. | The run may have failed at retrieval, primary-antibody binding or detection; the cause is undetermined from the slide alone (general IHC practice). Pancreatic endocrine cells are reported High (HPA: pancreas). | Confirm that endocrine cells are present, then check the run controls and optimize retrieval and antibody conditions within the assay's validated range (general IHC practice). Reassess study negatives only after the positive control works. |
| Esophageal squamous cells stain as strongly as the chosen positive control. | That conflicts with the HPA Not detected observation (HPA: esophagus). Antibody cross-reactivity or endogenous detection activity may contribute (general IHC practice). | Review the no-primary control, compare staining compartments and confirm the expected positive-cell pattern in the same run (general IHC practice; HPA: tissue IHC). Treat the discordant staining as unresolved until controls clarify it. |
| A strong signal appears only along plasma membranes. | A membrane-only distribution conflicts with ACLY's cytosolic annotation and lack of a transmembrane segment (UniProt P53396: subcellular location; topology). | Check tissue morphology, the no-primary control and whether cytoplasmic signal is present in a reported High cell population (general IHC practice; HPA: tissue IHC). Avoid scoring membrane-only color as confirmed ACLY. |
| Nuclei stain while cytoplasm is weak. | Nuclear tissue staining has been observed (HPA: tissue IHC), although cytosol is ACLY's UniProt location (UniProt P53396: subcellular location). The available evidence does not make nuclear-only staining diagnostic. | Compare the cell type and compartment with HPA tissue observations, then inspect positive and no-primary controls under the same run conditions (HPA: tissue IHC; general IHC practice). Record the nuclear-only pattern separately from an expected cytoplasmic result. |
| Diffuse chromogen obscures differences between cells. | Excess nonspecific signal or detection-system activity may obscure localization (general IHC practice); HPA staining levels do not establish the cause (HPA: tissue IHC). | Inspect the no-primary control and review blocking, washing and chromogen development; repeat with settings that preserve the positive-control cell pattern (general IHC practice; HPA: tissue IHC). |
| A Medium or Not detected HPA population gives a different result in the study specimen. | HPA reports cell-specific reference observations and notes low consistency between staining and RNA expression (HPA: tissue IHC; reliability description). A discrepant specimen alone cannot identify a technical or biological cause. | Verify the cell identity and run controls, document the compartment and intensity, and interpret the discrepancy against the relevant HPA cell-level observation (general IHC practice; HPA: tissue IHC). |
Experiment evidence
IHC & IF images showing ACLY staining
PB10024 · IHCIHC analysis of ACLY using anti-ACLY antibody (PB10024). ACLY was detected in a paraffin-embedded section of human colonic adenocarcinoma ti
PB10024 · IHCIHC analysis of ACLY using anti-ACLY antibody (PB10024). ACLY was detected in a paraffin-embedded section of human hepatocellular carcinoma
PB10024 · IFIF analysis of ACLY using anti-ACLY antibody (PB10024). ACLY was detected in an immunocytochemical section of A594 cells. Enzyme antigen ret
M02372-1 · IHCIHC analysis of ATP citrate lyase using anti-ATP citrate lyase antibody (M02372-1). ATP citrate lyase was detected in paraffin-embedded sect
M02372-1 · IHCIHC analysis of ATP citrate lyase using anti-ATP citrate lyase antibody (M02372-1). ATP citrate lyase was detected in paraffin-embedded sect
M02372-1 · IFIF analysis of ATP citrate lyase using anti-ATP citrate lyase antibody (M02372-1). ATP citrate lyase was detected in immunocytochemical sect
M02372 · IHCImmunohistochemical analysis of paraffin-embedded human brain carcinoma, using ATP citrate lyase Antibody .
M02372-3 · IHCHuman hepatocellular carcinoma was stained with anti-ATP-Citrate Lyase rabbit antibody
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000131473-ACLY/tissue/cerebral+cortex#imid_5860408
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000131473-ACLY/tissue/colon#imid_5860334
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000131473-ACLY/tissue/liver#imid_5860335
HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000131473-ACLY/tissue/pancreas#imid_5860286
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA022434; green = ACLY, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA022434; green = ACLY, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ACLY IHC & IF
🧪Run pancreas first and expect staining in pancreatic endocrine cells (HPA: High in pancreatic endocrine cells). Use esophageal squamous epithelial cells as the negative tissue (HPA: Not detected); on the pancreatic slide, treat cells without specific cytosolic staining as internal background references, without assuming a named neighboring cell type is ACLY-negative (UniProt P53396: cytosol).
Positive control tissue: Pancreas (Pancreatic endocrine cells, HPA High)
Negative control tissue: Esophagus (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ACLY in A-431, U-251MG, U2OS, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), Sperm, with annotated localisation: Cytosol (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; a concentration-matched rabbit IgG isotype control (selected PB10024 caption: rabbit primary); and ACLY-knockout material or a validated peptide-block control. For pancreatic chromogenic IHC, block endogenous peroxidase and check for residual background before scoring DAB staining (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected PB10024 paraffin-section caption does not state the fixative (selected PB10024 caption). Heat retrieval in EDTA at pH 8.0 is a documented starting condition, but retrieval dependence has not been established; neither frozen sections nor IF/ICC is shown to be easier for ACLY in the supplied evidence (selected PB10024 caption). Locate pancreatic endocrine cells carefully when scoring, because the expected positive population is cell-type specific (HPA: High in pancreatic endocrine cells).
HPA tissue IHC evidence for ACLY
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Low 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 |
|---|---|---|---|---|
| Pancreas | Pancreatic endocrine cells | High | Protein (IHC) | HPA → |
| Prostate | Glandular cells | High | Protein (IHC) | HPA → |
| Testis | Leydig cells | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | Cells in molecular layer | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Fallopian tube | Ciliated cells (cell body) | Not detected | Protein (IHC) | HPA → |
| Oral mucosa | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Ovary | Ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ACLY IHC Tips
Troubleshoot ACLY staining in paraffin sections by checking retrieval, compartment, cell type and controls before comparing signal intensity (UniProt P53396; HPA tissue IHC).
Which retrieval conditions should I try first for weak ACLY staining in paraffin sections?
Start with heat-mediated retrieval in EDTA pH 8.0 for paraffin sections stained with PB10024 (datasheet PB10024). The demonstrated tissue workflow then used 10% goat serum block and 2 μg/mL primary antibody overnight at 4°C (PB10024 tissue-IHC caption). If signal remains weak, compare retrieval duration on adjacent sections while keeping antibody concentration, development time and section thickness constant (standard IHC practice). Excessive heating can damage tissue morphology or increase nonspecific staining, so judge any gain in cytoplasmic signal alongside intact architecture and a matched negative control (standard IHC practice; UniProt P53396 localisation).
How should I investigate weak ACLY staining after fixation?
The PB10024 tissue caption describes a paraffin section but does not state its fixative; ACLY-specific sensitivity to fixation is therefore unknown (PB10024 tissue-IHC caption). Record the fixative, fixation duration and processing history for each specimen before comparing staining intensity across sections (standard IHC practice). Test sections with different documented fixation histories using the same EDTA pH 8.0 retrieval and 2 μg/mL primary antibody conditions (datasheet PB10024; PB10024 tissue-IHC caption). Compare preserved morphology and cytoplasmic staining in the relevant cells, because fixation differences and tissue damage can both alter apparent signal (standard IHC practice; UniProt P53396 localisation).
Should nuclear ACLY staining be considered specific in chromogenic IHC?
Expect a substantial cytoplasmic component: ACLY is annotated in the cytosol, and its sequence has no transmembrane segment (UniProt P53396 localisation and topology). Tissue IHC reports cytoplasmic and nuclear staining, although its antibody staining has low consistency with RNA expression (HPA tissue IHC). Mainly cytosolic localisation is supported by subcellular imaging, whereas nucleoplasmic localisation is uncertain (HPA subcellular). Score nuclear and cytoplasmic staining separately, compare their distribution with cell morphology, and seek independent antibody or orthogonal evidence before treating nuclear-only DAB signal as ACLY (standard IHC practice; HPA subcellular).
Could an epitope or isoform difference explain inconsistent ACLY staining?
ACLY has 3 annotated isoforms, but the supplied PB10024 caption does not identify its binding epitope or establish isoform coverage (UniProt P53396 isoforms; PB10024 tissue-IHC caption). The protein has documented modified residues, including phosphorylation at positions 263, 447, 451 and 455 (UniProt P53396 modified residues). Do not assign a staining difference to one isoform or modification without epitope mapping and appropriate validation (standard IHC practice). First compare adjacent sections under matched retrieval and detection conditions, then use an independently characterised antibody if an epitope-dependent explanation remains plausible (standard IHC practice).
How can I assess ACLY by IF alongside a cell-type marker?
For a separate IF experiment, pair ACLY with a marker that identifies the pancreatic endocrine cells being assessed; those cells have high tissue-IHC expression in the supplied profile (HPA tissue IHC). Choose spectrally separated fluorophores and place the weaker signal in a channel with low measured tissue autofluorescence, using single-stain and unstained controls (standard IF practice). ACLY is cytosolic and has no transmembrane segment, so permeabilise the cell membrane for access to intracellular epitopes while checking that morphology remains intact (UniProt P53396 localisation and topology; standard IF practice). Validate the antibody for IF independently of the PB10024 paraffin-section IHC example (PB10024 tissue-IHC caption; standard IF practice).
What should I check when ACLY DAB staining appears widespread?
Compare the stained section with a matched section lacking primary antibody to identify signal from detection reagents or endogenous peroxidase (standard IHC practice). Include a peroxidase block before DAB development and check whether background follows tissue edges, damaged areas or residual reagent pools (standard IHC practice). The PB10024 example used 10% goat serum block, 2 μg/mL primary antibody overnight at 4°C, and a peroxidase-conjugated secondary for 30 minutes at 37°C (PB10024 tissue-IHC caption). Widespread staining alone is inconclusive because ACLY is cytosolic and HPA reports expression across several tissues (UniProt P53396 localisation; HPA tissue IHC).
How should I quantify ACLY staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region of interest before scoring, then record cytoplasmic intensity and the percentage of positive cells as an H-score (standard IHC practice; UniProt P53396 localisation). Score nuclear staining separately because tissue IHC reports it while subcellular evidence rates nucleoplasmic localisation as uncertain (HPA tissue IHC; HPA subcellular). Normalise positive-cell counts to the total number of evaluable cells in the same compartment, or report positive-cell density per mm² of viable tissue (standard IHC practice). Keep retrieval, staining batch, imaging settings and scoring thresholds matched, and exclude folds, necrosis and section edges from analysis (standard IHC practice).
How can I distinguish credible ACLY staining from artefact?
A credible pattern includes cytoplasmic staining in intact cells, consistent with ACLY's supported cytosolic localisation (UniProt P53396 localisation; HPA subcellular). Pancreatic endocrine cells, prostate glandular cells and testicular Leydig cells are reported as high-staining examples, while several other listed cell populations are not detected (HPA tissue IHC). Treat isolated nuclear staining cautiously because nucleoplasmic localisation remains uncertain, and inspect edge effects, folds and necrotic areas for uneven DAB deposition (HPA subcellular; standard IHC practice). Use a primary-omission control to assess endogenous enzyme or detection background, and corroborate unexpected cell-type patterns independently because tissue-IHC staining shows low consistency with RNA expression (standard IHC practice; HPA tissue IHC).
Boster reagents
Best ACLY / ATP-citrate synthase IHC Antibodies
Anti-ACLY antibodies have IHC images from human tissues and mouse and rat pancreas, plus IF/ICC images from MCF7 and A594 cells (catalog image captions: M02372-1, PB10024).
Real IHC data
Anti-ATP citrate lyase/ACLY Antibody ®
Cat # PB10024
Real IHC data
Anti-ATP citrate lyase ACLY Antibody ® (monoclonal, 5I2)
Cat # M02372-1
Real IHC data
Anti-ATP citrate lyase ACLY Rabbit Monoclonal Antibody
Cat # M02372
Real IHC data
Anti-ATP-Citrate Lyase Rabbit Monoclonal Antibody
Cat # M02372-3
PB10024 shows paraffin-section IHC in human colonic adenocarcinoma, hepatocellular carcinoma, laryngeal squamous cell carcinoma and placenta, plus IF in A594 cells (catalog image captions: PB10024); M02372-1 shows paraffin-section IHC in human pancreatic and testis cancer and mouse and rat pancreas, plus IF in MCF7 cells (catalog image captions: M02372-1). M02372 shows paraffin-section IHC in human brain carcinoma (catalog image caption: M02372); M02372-3 shows IHC staining of human hepatocellular carcinoma, with section processing unreported (catalog image caption: M02372-3).
Which to pick: For paraffin-section IHC, consider PB10024, a rabbit antibody listed at 1–2 μg/ml, or M02372-1, a mouse monoclonal listed at 0.5–1 μg/ml; each antibody’s own IHC caption documents EDTA pH 8 retrieval and DAB detection, but neither reports the fixative (catalog host/dilutions and IHC image captions: PB10024, M02372-1). For IF/ICC, M02372-1 has an MCF7 cell image and a listed dilution of 2 μg/ml, while PB10024 has an A594 cell image and a listed dilution of 5 μg/ml (catalog IF image captions/dilutions: M02372-1, PB10024). For cross-species tissue IHC, prioritize M02372-1: its own IHC captions show human, mouse and rat samples, matching its listed reactivity (catalog IHC image captions/reactivity: M02372-1).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P53396 (ACLY_HUMAN, ATP-citrate synthase).
- Human Protein Atlas. ACLY tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. ACLY subcellular location (ICC-IF): Mainly localized to the cytosol. In addition localized to the nucleoplasm, primary cilium transition zone, basal body and principal piece..
- Human Protein Atlas. ACLY antibody validation summary (5 antibodies).
- A novel metabolic function of Myc in regulation of fatty acid synthesis in prostate cancer. Oncogene 2021 — PMC7855479.
- Prognostic Value of Malic Enzyme and ATP-Citrate Lyase in Non-Small Cell Lung Cancer of the Young and the Elderly. PloS one 2015 — PMC4427316.
- Role of ACLY in the development of gastric cancer under hyperglycemic conditions. Quantitative biology (Beijing, China) 2024 — PMC12806462.
- Leelamine Is a Novel Lipogenesis Inhibitor in Prostate Cancer Cells In Vitro and In Vivo. Molecular cancer therapeutics 2019 — PMC6774899.
- PubMed PMID:1371749 — UniProt-cited evidence.
- PubMed PMID:9116495 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ACLY (ATP-citrate synthase). Expected localisation Cytoplasmic and nuclear tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PB10024). Validated IHC antibody: PB10024 at 1-2μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Pancreas (Pancreatic endocrine cells); negative Esophagus. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colonic adenocarcinoma tissue; fixative not specified (datasheet PB10024); 2. Fixation: Image fixative and duration unreported (datasheet PB10024); 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 PB10024); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB10024); 8. Primary antibody: Rabbit anti-ACLY, 1-2μg/ml (datasheet PB10024); 9. Primary incubation: Overnight at 4 °C (datasheet PB10024). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific). Selected-image fixative and duration unreported (datasheet PB10024); verify before use.; Caveat: Antibody staining has low concordance with RNA expression (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope coverage is unresolved (UniProt); Regulation: No expression regulator annotated (UniProt). Evidence: UniProt P53396, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the cytosol. In addition localized to the…, Datasheet PB10024, PMC4427316, PMC6774899. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ACLY/immunohistochemistry-ihc-guide.