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ACSL4 / Long-chain-fatty-acid--CoA ligase 4 · IHC design guide
Design Immunohistochemistry for ACSL4
Plan chromogenic ACSL4 IHC in paraffin sections around the observed cytoplasmic tissue staining (HPA tissue IHC). Cortical and hippocampal neurons and alveolar type II cells stain strongly, while bone marrow hematopoietic cells are not detected (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ACSL4 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 IHC; ER and outer mitochondrial membrane locations annotated (HPA tissue IHC; UniProt) | |
| Staining pattern | Cytoplasmic staining across tissues; high in cortical neurons (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M04372) | |
| Positive control | Cerebral cortex+4 more · see all | |
| Negative control | Bone marrow+1 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) | |
| Caveat | Hematopoietic cells may be unstained despite broad tissue staining (HPA tissue IHC) | |
| Regulation | Expression regulation unreported (UniProt) | |
| Isoform / epitope | 2 isoforms; cytoplasmic region 29–711; epitope unknown (UniProt) |
Section 1
Recommended ACSL4 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is followed by published ACSL4 staining methods for pancreatic lesions and liver tissue microarrays (PMC12065365; PMC7198044).
| Sample | Paraffin-embedded human bladder epithelial carcinoma tissue; fixative not specified (datasheet M04372) |
| Fixation | Image fixative and duration unreported (datasheet M04372); 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 M04372); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M04372) |
| Primary antibody | Mouse monoclonal (clone 4I7) anti-ACSL4, 2 μg/ml (datasheet M04372) |
| Primary incubation | Overnight at 4 °C (datasheet M04372) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet M04372) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ACSL4-positive staining in neuronal cells of cerebral cortex (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet M04372); the pancreatic study used pH 9.0 retrieval with a different antibody (PMC12065365).
Section 2
What Is the Expected ACSL4 Staining Pattern?
ACSL4 should appear predominantly cytoplasmic in IHC, with stronger staining expected in cerebral cortex and hippocampal neurons and lung alveolar type II cells (HPA: ubiquitous cytoplasmic expression; High in those cells). UniProt places ACSL4 at the mitochondrial outer membrane, endoplasmic reticulum membrane and cell membrane, with residues 29–711 facing the cytoplasm (UniProt O60488: localization and topology). Interpret modest staining cautiously because tissue IHC reliability is Supported, with medium consistency against RNA data (HPA: tissue IHC).
| Cytoplasmic staining in neuronal cells of cerebral cortex or hippocampus, or alveolar type II cells in lung. | This fits the strongest supplied tissue examples (HPA: High in each listed cell type). Judge the pattern in the named cells rather than treating every cell in the section as an equivalent positive control (HPA: cell-specific tissue IHC). |
| A predominantly nuclear pattern, especially when cytoplasm is clear. | Treat this as discordant with the reported tissue pattern (HPA: ubiquitous cytoplasmic expression) and with the annotated membrane-associated, cytoplasm-facing protein (UniProt O60488: localization and topology). Check controls and staining conditions before assigning it to ACSL4. |
| Strong staining in bone-marrow hematopoietic cells or ovarian stromal cells. | These cells were not detected in the supplied tissue IHC data (HPA: bone marrow and ovary). Cross-reactivity or endogenous detection activity are possibilities, not diagnoses from appearance alone. Compare a no-primary control and review the cell identity. |
| Widespread, diffuse chromogen obscures cell borders and tissue structure. | A diffuse haze cannot establish the reported cytoplasmic pattern (HPA: tissue IHC profile). General IHC practice is to assess a no-primary control, blocking, washes and detection background, then reassess whether signal is confined to identifiable cells. |
| No convincing signal in the expected positive cells of a cortex, hippocampus or lung section. | This conflicts with their reported High staining (HPA: neuronal cells; alveolar type II cells). First confirm the intended cells are present and inspect a same-run positive control; weak detection or staining conditions remain possible until controls are reviewed. |
💡Expected ACSL4 appearanceCall an IHC result positive when identifiable cortex or hippocampal neurons, or lung alveolar type II cells, show clear, relatively strong cytoplasmic staining (HPA: High; cytoplasmic profile); predominantly nuclear staining or signal concentrated in HPA-undetected cell types warrants investigation (HPA: tissue IHC; UniProt O60488: topology).
| Compartment and topology | ACSL4 is annotated at mitochondrial outer, endoplasmic reticulum and cell membranes, with a cytoplasmic region spanning residues 29–711 (UniProt O60488: localization and topology). At light-microscope IHC resolution, use the observed cytoplasmic pattern as the practical readout (HPA: tissue IHC profile). |
| Strength of tissue evidence | The tissue IHC profile is Supported, with medium consistency between antibody staining and RNA expression (HPA: reliability). A matching pattern supports interpretation, but this rating does not establish that every stained structure is specific. |
| Cell-dependent signal range | HPA reports High staining in selected neurons and alveolar type II cells, Medium in adipocytes and several other listed cells, and no detection in bone-marrow hematopoietic or ovarian stromal cells (HPA: tissue IHC). Score the relevant cell population rather than whole-section darkness. |
| Isoforms and epitope | Long and Short isoforms are annotated (UniProt O60488: isoforms). The supplied sources give no antibody epitope or isoform-recognition data, so neither a difference between isoforms nor isoform-specific staining can be inferred from this section. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive cells are pale or blank. | The result disagrees with reported High staining in cortex or hippocampal neurons and lung alveolar type II cells (HPA: tissue IHC); the slide alone cannot identify the failed step. | Confirm those cells are present, then compare a same-run positive control. As general IHC practice, review antigen retrieval, antibody dilution and detection performance before interpreting absence as biological. |
| Nuclei dominate the signal. | This differs from the reported cytoplasmic tissue profile (HPA: tissue IHC) and cytoplasm-facing topology (UniProt O60488: topology). | Check the no-primary control and whether nuclear color persists without primary antibody. Reassess localization in intact cells; do not score a nuclear-only result as the expected ACSL4 pattern. |
| Undetected-reference cells stain strongly. | Bone-marrow hematopoietic and ovarian stromal cells were not detected in the supplied HPA examples (HPA: tissue IHC). Cross-reactivity and endogenous detection activity remain possible explanations. | Verify the stained cell population, compare no-primary and positive controls, and inspect the detection background. Treat the HPA examples as reference observations rather than a universal absence claim. |
| Diffuse color makes intensity hard to score. | Background can obscure the cell-localized cytoplasmic profile (HPA: tissue IHC profile); appearance alone does not identify which reagent or step caused it. | Use the no-primary control to assess detection background. As general IHC practice, review blocking, washes and chromogen development, then score only interpretable cells. |
| A cytoplasmic signal appears weaker in some tissue cells. | The supplied HPA observations span High, Medium, Low and Not detected across named cell populations (HPA: tissue IHC). Differences in whole-section intensity therefore need cell-level context. | Record the tissue and cell type before comparing slides. Use the same-run controls and consistent scoring criteria; do not convert a lower HPA category into a technical failure without further evidence. |
| How should an IF/ICC pattern be compared with this IHC result? | HPA reports mainly Golgi localization with additional mitochondrial localization in ICC-IF (HPA: approved subcellular locations), while tissue IHC reports a broadly cytoplasmic profile (HPA: tissue IHC). | Interpret each result in its assay context. The finer ICC-IF localization can inform review of an image, but it does not make a Golgi-shaped pattern a required criterion for chromogenic tissue IHC (HPA: subcellular and tissue profiles). |
Experiment evidence
IHC & IF images showing ACSL4 staining
M04372 · IHCIHC analysis of FACL4/ACSL4 using anti-FACL4/ACSL4 antibody (M04372). FACL4/ACSL4 was detected in a paraffin-embedded section of human bladd
M04372 · IHCIHC analysis of FACL4/ACSL4 using anti-FACL4/ACSL4 antibody (M04372). FACL4/ACSL4 was detected in a paraffin-embedded section of human bladd
M04372 · IFIF analysis of FACL4/ACSL4 using anti-FACL4/ACSL4 antibody (M04372). FACL4/ACSL4 was detected in an immunocytochemical section of SiHa cells
A04372-2 · IHCIHC analysis of FACL4/ACSL4 using anti-FACL4/ACSL4 antibody (A04372-2). FACL4/ACSL4 was detected in a paraffin-embedded section of mouse bra
A04372-2 · IHCIHC analysis of FACL4/ACSL4 using anti-FACL4/ACSL4 antibody (A04372-2). FACL4/ACSL4 was detected in a paraffin-embedded section of rat brain
A04372-2 · IFIF analysis of FACL4/ACSL4 using anti-FACL4/ACSL4 antibody (A04372-2). FACL4/ACSL4 was detected in an immunocytochemical section of A431 cel
M04372-2 · IHCRat liver was stained with anti-ACSL4 rabbit antibody
M04372-2 · IHCHuman hepatocellular carcinoma was stained with anti-ACSL4 rabbit antibody
M04372-1 · IFImmunofluorescent analysis using the Antibody at 1:50 dilution.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000068366-ACSL4/tissue/cerebral+cortex#imid_1798208
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000068366-ACSL4/tissue/colon#imid_1798126
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000068366-ACSL4/tissue/liver#imid_1798135
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000068366-ACSL4/tissue/kidney#imid_1798136
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA000286; green = ACSL4, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA000286; green = ACSL4, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ACSL4 IHC & IF
🧪Start with lung; alveolar type II cells should stain (HPA: High in alveolar type II cells). Use bone marrow hematopoietic cells as the negative tissue (HPA: Not detected in hematopoietic cells); within the lung section, cells without specific staining should show only background, but HPA does not identify a validated negative cell population there (HPA: lung row).
Positive control tissue: Cerebral cortex (Neuronal cells, HPA High)
Negative control tissue: Bone marrow (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ACSL4 in A-431, U-251MG, U2OS, with annotated localisation: Golgi apparatus (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a host-species-matched isotype control; use ACSL4 knockout tissue as a biological specificity control if available (standard IHC practice). Quench endogenous peroxidase for chromogenic detection and assess lung background, including pigment in alveolar macrophages; for IF, assess tissue autofluorescence (standard IHC/IF practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected M04372 paraffin-section caption does not state the fixative (M04372 tissue-IHC caption). That caption uses heat retrieval in EDTA, pH 8.0, but does not establish whether ACSL4 staining depends on retrieval (M04372 tissue-IHC caption). ICC-IF images exist for A-431, U-251MG and U2OS (HPA: subcellular images); whether IF or frozen sections are easier than paraffin IHC is unreported, and lung pigment can complicate chromogenic interpretation (standard IHC practice).
HPA tissue IHC evidence for ACSL4
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — 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 |
|---|---|---|---|---|
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Hippocampus | Neuronal cells | High | Protein (IHC) | HPA → |
| Lung | Alveolar cells type II | High | Protein (IHC) | HPA → |
| Adipose tissue | Adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced ACSL4 IHC Tips
Troubleshoot ACSL4 staining in paraffin sections by checking retrieval, cell type, subcellular pattern and controls before interpreting chromogenic signal.
Which retrieval conditions should I try first for ACSL4 in paraffin sections?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for ACSL4 paraffin sections (datasheet M04372). Keep section thickness, heating and cooling conditions consistent while comparing runs, because these variables affect IHC signal (standard IHC practice). The documented staining used 2 μg/ml primary antibody overnight at 4°C, so reproduce those conditions before changing several variables together (datasheet M04372). If staining remains weak, test a retrieval adjustment on matched sections alongside the documented condition, and judge improvement by cellular localisation and background rather than intensity alone (standard IHC practice; UniProt O60488 localisation).
Could fixation explain weak or uneven ACSL4 staining?
ACSL4-specific sensitivity to fixation is unknown here; the selected paraffin-section caption does not state a fixative (datasheet M04372). Record fixation method and duration for each specimen, then compare similarly processed sections when assessing weak or uneven chromogenic staining (standard IHC practice). Excessive or inconsistent fixation can alter antigen accessibility in IHC generally, but that observation does not establish an ACSL4-specific effect (standard IHC practice). Keep the documented EDTA retrieval at pH 8.0 and 2 μg/ml overnight primary incubation constant while assessing the fixation variable (datasheet M04372).
What subcellular ACSL4 pattern should I expect in chromogenic IHC?
Expect a predominantly cytoplasmic pattern in tissue IHC, while allowing for membrane-associated staining that chromogenic sections may not resolve into separate organelles (HPA tissue IHC; UniProt O60488 localisation). ACSL4 is annotated at the mitochondrial outer membrane, endoplasmic reticulum membrane and cell membrane, with residues 29–711 facing the cytoplasm (UniProt O60488 topology). HPA cell imaging instead identifies the Golgi apparatus as its main location and mitochondria as an additional location (HPA subcellular). Judge a proposed compartment pattern against the section's morphology and controls; a diffuse nuclear-only pattern would conflict with these reported locations (standard IHC practice; UniProt O60488 localisation; HPA subcellular).
Can isoforms or epitope placement change how I interpret ACSL4 staining?
ACSL4 has Long and Short isoforms, but the supplied antibody caption does not identify its epitope or establish recognition of both isoforms (UniProt O60488 isoforms; datasheet M04372). Its single transmembrane segment spans residues 8–28, leaving residues 29–711 on the cytoplasmic side (UniProt O60488 topology). Phosphorylation is annotated at residues 328, 447 and 679; whether any modification changes this antibody's IHC binding is unknown (UniProt O60488 modified residues; datasheet M04372). If staining differs across samples, check antibody epitope documentation and use an independent specificity control before assigning that difference to an isoform or modification (standard IHC practice).
How should I assess ACSL4 by IF alongside this IHC workflow?
Treat IF as a separate assay: the documented 2 μg/ml overnight incubation and EDTA retrieval at pH 8.0 belong to the catalog antibody's paraffin IHC example (datasheet M04372). For multiplex IF, pair ACSL4 with a marker for the cell type under study; HPA reports high tissue-IHC staining in neuronal cells and lung alveolar type II cells (HPA tissue IHC; standard IF practice). Choose fluorophores after examining tissue autofluorescence and include single-stain controls to assess spectral overlap (standard IF practice). If targeting an epitope within cytoplasmic residues 29–711, optimise permeabilisation for access across membranes, while recognising that this antibody's epitope is unspecified (UniProt O60488 topology; datasheet M04372; standard IF practice).
How do I distinguish ACSL4 staining from chromogenic background?
The documented paraffin IHC example used 10% goat serum blocking, a biotinylated secondary, a streptavidin–biotin complex and DAB detection (datasheet M04372). Include a no-primary control and apply a peroxidase block when using DAB to assess nonspecific or endogenous enzyme signal (standard IHC practice). With a biotin-based system, check whether tissue-dependent endogenous biotin contributes to staining, especially when signal persists without primary antibody (standard IHC practice; datasheet M04372 detection method). Evaluate signal in intact cells and expected cytoplasmic regions, then adjust blocking, antibody concentration or detection conditions one variable at a time (HPA tissue IHC; standard IHC practice).
How should I quantify ACSL4 across paraffin tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored cell population and compartment before measuring ACSL4, because HPA describes broadly cytoplasmic tissue expression with cell-type variation (HPA tissue IHC). For chromogenic sections, report an H-score or the percentage of positive cells with intensity categories, using the same thresholds across cases (standard IHC practice). Normalise positive-cell counts to the number of eligible cells, or report positive-cell density per mm² of viable tissue when cell counting is impractical (standard IHC practice). Exclude folds, edges and necrotic regions by a prespecified rule, and compare sections stained under matched retrieval and detection conditions (standard IHC practice).
When is an ACSL4-positive IHC result biologically credible?
A credible result follows cellular morphology and a predominantly cytoplasmic pattern, consistent with HPA tissue IHC and ACSL4's annotated membrane-associated locations (HPA tissue IHC; UniProt O60488 localisation). Interpret cell identity explicitly: HPA reports high staining in neuronal cells and lung alveolar type II cells, while bone-marrow hematopoietic cells were not detected in its tissue survey (HPA tissue IHC). Nuclear-only staining, section-edge accentuation or signal concentrated in necrotic material warrants review against controls and intact tissue (UniProt O60488 localisation; standard IHC practice). Persistent DAB signal in a no-primary control raises concern for endogenous enzyme or detection background rather than specific ACSL4 staining (standard IHC practice).
Boster reagents
Best ACSL4 / Long-chain-fatty-acid--CoA ligase 4 IHC Antibodies
Anti-ACSL4 antibodies have IHC images from human, mouse, and rat tissues, plus IF images from cultured cells and an unspecified sample (catalog image captions).
Real IHC data
Anti-FACL4/ACSL4 Antibody ® (monoclonal, 4I7)
Cat # M04372
Real IHC data
Anti-FACL4/ACSL4 Antibody ®
Cat # A04372-2
Real IHC data
Anti-ACSL4 Rabbit Monoclonal Antibody
Cat # M04372-2
Real IF data
Anti-FACL4 Rabbit Monoclonal Antibody
Cat # M04372-1
M04372 has IHC images from human bladder, lung, and renal pelvis specimens; A04372-2 has IHC images from mouse and rat brain and human liver cancer, plus IF data from A431 cells (catalog image captions). M04372-2 has IHC images from rat and mouse liver and human hepatocellular carcinoma; M04372-1 lists IHC and IF applications, with an IF image whose sample is unspecified (catalog applications and image captions).
Which to pick: For human tissue IHC, choose M04372, a mouse monoclonal with a human paraffin-section image; for mouse or rat paraffin sections, choose A04372-2, a rabbit antibody with brain-section images from both species (catalog host, reactivity, and each SKU’s IHC image captions). For IF/ICC, A04372-2 has an A431 cell image, while M04372-1 has an IF image without a stated sample identity (catalog IF image captions). For cross-species tissue IHC, M04372-2 has human, mouse, and rat reactivity and IHC images from all three species; its captions do not report section processing, and the IHC captions for these antibodies do not report the fixative (catalog reactivity and IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O60488 (ACSL4_HUMAN, Long-chain-fatty-acid--CoA ligase 4).
- Human Protein Atlas. ACSL4 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. ACSL4 subcellular location (ICC-IF): Mainly localized to the Golgi apparatus. In addition localized to the mitochondria..
- Human Protein Atlas. ACSL4 antibody validation summary (2 antibodies).
- Scutellaria barbata Inhibits Hepatocellular Carcinoma Tumorigenicity by Inducing Ferroptosis of Hepatocellular Carcinoma Cells. Frontiers in oncology 2022 — PMC8936062.
- A CoQ10 analog ameliorates cognitive impairment and early brain injury after subarachnoid hemorrhage by regulating ferroptosis and neuroinflammation. Redox biology 2025 — PMC12148479.
- Long-chain fatty acyl CoA synthetase 4 expression in pancreatic cancer: a marker for malignant lesions and prognostic indicator for recurrence. Diagnostic pathology 2025 — PMC12065365.
- Immunohistochemical staining reveals differential expression of ACSL3 and ACSL4 in hepatocellular carcinoma and hepatic gastrointestinal metastases. Bioscience reports 2020 — PMC7198044.
- PubMed PMID:9598324 — UniProt-cited evidence.
- PubMed PMID:9480748 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ACSL4 (Long-chain-fatty-acid--CoA ligase 4). Expected localisation Cytoplasmic IHC; ER and outer mitochondrial membrane locations annotated (HPA tissue IHC; UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M04372). Validated IHC antibody: M04372 at 2 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Cerebral cortex (Neuronal cells); negative Bone marrow. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human bladder epithelial carcinoma tissue; fixative not specified (datasheet M04372); 2. Fixation: Image fixative and duration unreported (datasheet M04372); 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 M04372); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M04372); 8. Primary antibody: Mouse monoclonal (clone 4I7) anti-ACSL4, 2 μg/ml (datasheet M04372); 9. Primary incubation: Overnight at 4 °C (datasheet M04372). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Hematopoietic cells may be unstained despite broad tissue staining (HPA tissue IHC); Isoform / epitope: 2 isoforms; cytoplasmic region 29–711; epitope unknown (UniProt); Regulation: Expression regulation unreported (UniProt). Evidence: UniProt O60488, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the Golgi apparatus. In addition localized to…, Datasheet M04372, PMC12065365, PMC7198044. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ACSL4/immunohistochemistry-ihc-guide.