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ACSL6 / Long-chain-fatty-acid--CoA ligase 6 · IHC design guide
Design Immunohistochemistry for ACSL6
Plan chromogenic ACSL6 IHC in paraffin sections using seminal vesicle glandular cells or late spermatids as positive controls (HPA tissue IHC). This guide covers fixation consistency, antibody titration and scoring of cytoplasmic staining (standard IHC practice; HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ACSL6 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 | Cytoplasm observed; organelle membranes annotated (HPA tissue IHC; UniProt) | |
| Staining pattern | Cytoplasmic signal in glandular cells and late spermatids (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Seminal vesicle+4 more · see all | |
| Negative control | Bone marrow+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 | Medium consistency between staining and RNA data (HPA tissue IHC) | |
| Regulation | Staining regulation unreported (UniProt) | |
| Isoform / epitope | 9 isoforms; cytoplasmic region aa 46–697 (UniProt) |
Section 1
Recommended ACSL6 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published ACSL6 staining details for liver cancer specimens (PMC11409972) and paraffin sections (PMC11759416).
| Sample | Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A07813-2) |
| Fixation | Image fixative and duration unreported (datasheet A07813-2); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-ACSL6, 1:50-1:200 (datasheet A07813-2) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ACSL6-positive staining in glandular cells of seminal vesicle (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression mainly in testis, seminal vesicle, small intestines and brain. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page antigen-retrieval rule: cytoplasmic/membrane antigen). Neither included excerpt specifies retrieval.
Section 2
What Is the Expected ACSL6 Staining Pattern?
ACSL6 is a membrane-associated enzyme with a cytoplasmic domain; UniProt places it at mitochondrial outer, peroxisomal, microsomal and endoplasmic reticulum membranes (UniProt Q9UKU0 topology and subcellular location). In tissue IHC, expect cytoplasmic staining, especially in seminal vesicle and small-intestine glandular cells and testicular late spermatids (HPA tissue IHC: High). HPA rates its tissue staining Enhanced, with medium agreement between staining and RNA data; external verification remains pending (HPA tissue IHC).
| Cytoplasmic staining in seminal vesicle or small-intestine glandular cells, or testicular late spermatids. | This matches HPA's strongest reported tissue IHC pattern (HPA tissue IHC: High in these cells). Assess the named cells against neighboring cells on the same section; a uniformly stained field gives less confidence in cell-specific signal (standard IHC interpretation). |
| Predominantly nuclear staining with little cytoplasmic signal. | This conflicts with the cytoplasmic tissue profile and membrane-associated UniProt locations (HPA tissue IHC; UniProt Q9UKU0 subcellular location). Treat an isolated nuclear pattern as suspect and compare it with a known-positive tissue and a no-primary control (standard IHC practice). |
| Strong signal in a cell type HPA reports as undetected, such as breast adipocytes or bronchial respiratory epithelium. | That pattern is discordant with the supplied tissue observations (HPA tissue IHC: Not detected in those cells). Cross-reactivity or endogenous detection activity are possibilities, not diagnoses; compare a no-primary control and repeat with an independently validated antibody (standard IHC practice). |
| Diffuse color across cells, stroma and section edges, obscuring cell boundaries. | The result cannot establish the cell-restricted cytoplasmic pattern reported by HPA (HPA tissue IHC). Uneven detection or background may be involved; inspect control slides and staining distribution before scoring ACSL6 expression (standard IHC interpretation). |
| No visible signal in a well-preserved seminal vesicle or small-intestine section. | This disagrees with High glandular-cell staining reported for those tissues (HPA tissue IHC). First verify that the expected glands are present; then check the IHC-validated antibody, detection reagents and run controls before interpreting the tissue as negative (standard IHC practice). |
💡Expected ACSL6 appearanceA convincing positive is cell-associated cytoplasmic staining, strongest in seminal vesicle or small-intestine glandular cells or testicular late spermatids (HPA tissue IHC: High); isolated nuclear color or indiscriminate staining of HPA-undetected cell types is suspect (HPA tissue IHC; UniProt Q9UKU0 subcellular location).
| Membrane topology | UniProt annotates one transmembrane segment at residues 25–45 and a cytoplasmic region at 46–697 (UniProt Q9UKU0 topology). This supports a cytoplasmic, membrane-associated interpretation but does not resolve individual organelles by chromogenic tissue IHC (standard IHC interpretation). |
| Tissue and cell selection | HPA reports High staining in seminal vesicle and small-intestine glandular cells and testicular late spermatids; cerebellar Purkinje cells and cortical glia are Medium (HPA tissue IHC). Select the specific cell population when judging a control, since an organ-wide score could hide a restricted pattern (standard IHC interpretation). |
| Bone marrow evidence differs by method | UniProt reports expression in erythrocyte precursors and bone marrow, while HPA tissue IHC lists bone-marrow hematopoietic cells as Not detected (UniProt Q9UKU0 tissue specificity; HPA tissue IHC). Do not treat marrow IHC as an assured positive control from these sources alone. |
| Antibody validation and isoforms | HPA lists antibody HPA040470 as IHC Enhanced and ICC Uncertain (HPA antibodies). UniProt lists 9 isoforms (UniProt Q9UKU0 isoforms); the supplied record gives no antibody epitope, so isoform coverage cannot be inferred. |
| IF/ICC: what appearance should I expect? | HPA's ICC-IF summary places ACSL6 mainly in vesicles, with additional cytosol localization marked uncertain; its main vesicle location is approved (HPA subcellular ICC-IF). This is an IF/ICC observation, not a required vesicle pattern for paraffin-section chromogenic IHC (HPA subcellular ICC-IF; HPA tissue IHC). |
| Fixation and retrieval evidence | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. |
| Situation | Likely cause | Next action |
|---|---|---|
| Strong nuclear staining dominates a nominal positive tissue. | The compartment conflicts with HPA's cytoplasmic tissue profile and UniProt's membrane locations (HPA tissue IHC; UniProt Q9UKU0 subcellular location). | Compare a known-positive section and no-primary control; score ACSL6 only where the expected cell-associated cytoplasmic pattern is supported (standard IHC practice). |
| A known-positive glandular tissue has no signal. | Absent staining conflicts with HPA's High glandular-cell observations, but does not identify which assay step failed (HPA tissue IHC). | Confirm glandular cells are present, inspect run controls and detection reagents, then optimize retrieval or antibody dilution using the IHC-validated antibody (standard IHC practice). |
| Brown color appears broadly, including stroma or slide edges. | Diffuse deposition can obscure HPA's cell-specific cytoplasmic pattern (HPA tissue IHC; standard IHC interpretation). | Review no-primary and reagent controls; check blocking, washing and chromogen development before assigning positive cells (standard IHC practice). |
| An HPA-undetected cell population stains strongly. | Cross-reactivity or endogenous chromogenic detection activity may explain discordant color; the HPA observation alone cannot distinguish them (HPA tissue IHC; standard IHC practice). | Compare the no-primary control, assess endogenous detection activity and seek an independent antibody result before reporting unexpected expression (standard IHC practice). |
| Bone marrow is negative despite an expectation from the UniProt tissue note. | The sources describe different evidence: UniProt reports expression in marrow-associated precursors, whereas HPA detects no staining in sampled hematopoietic cells (UniProt Q9UKU0 tissue specificity; HPA tissue IHC). | Use an HPA High tissue as the IHC positive control; describe a marrow result by its observed cell type and assay rather than treating the sources as equivalent (HPA tissue IHC; standard IHC interpretation). |
| An IF/ICC vesicle pattern seems different from broad cytoplasmic tissue IHC. | HPA reports approved vesicle localization in ICC-IF but a cytoplasmic tissue IHC profile; the assays resolve different features (HPA subcellular ICC-IF; HPA tissue IHC). | Interpret each assay against its own HPA reference and validation status; HPA lists ICC as Uncertain for HPA040470 (HPA antibodies; HPA subcellular ICC-IF). |
Experiment evidence
IHC & IF images showing ACSL6 staining
A07813-2 · IHCImmunohistochemistry (IHC) analyzes of ACSL6 (D531) pAb in paraffin-embedded human breast carcinoma tissue at 1:50.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000164398-ACSL6/tissue/cerebral+cortex#imid_20358309
HPA · CerebellumHuman Protein Atlas IHC, Cerebellum. Source: https://www.proteinatlas.org/ENSG00000164398-ACSL6/tissue/cerebellum#imid_20358320
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000164398-ACSL6/tissue/colon#imid_20361179
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000164398-ACSL6/tissue/liver#imid_20361188
HPA · ICC-IF U2OSHuman Protein Atlas immunofluorescence, U2OS, antibody HPA040470; green = ACSL6, blue = nucleus, red = microtubules.
HPA · ICC-IF U2OSHuman Protein Atlas immunofluorescence, U2OS, antibody HPA040470; green = ACSL6, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ACSL6 IHC & IF
🧪Run small intestine first: glandular cells should stain (HPA: High in small-intestine glandular cells). Use epididymis glandular cells as the negative tissue (HPA: Not detected in epididymis glandular cells); on the positive slide, assess morphologically distinct cells that remain unstained as an internal background reference, without assuming they are ACSL6-negative.
Positive control tissue: Seminal vesicle (Glandular cells, HPA High)
Negative control tissue: Bone marrow (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ACSL6 in U2OS, KOLF2.1J, with annotated localisation: Vesicles (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species and immunoglobulin class; use knockout material or a validated immunizing-peptide block as a biological specificity control (standard IHC practice). For chromogenic staining in small intestine, block endogenous peroxidase and, if using avidin–biotin detection, check for endogenous biotin signal (standard IHC practice).
⚠️Feasibility: The selected SKU A07813-2 caption shows staining in paraffin-embedded breast carcinoma at 1:50, but reports no fixative; a target-specific fixation window and retrieval dependence are unreported (selected-SKU IHC caption). The supplied evidence does not establish that frozen sections or IF are easier than paraffin IHC, although ICC-IF images exist for U2OS and KOLF2.1J (HPA subcellular). In small intestine, endogenous biotin can complicate avidin–biotin detection, so inspect the matched negative controls (standard IHC practice).
HPA tissue IHC evidence for ACSL6
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium 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 |
|---|---|---|---|---|
| Seminal vesicle | Glandular cells | High | Protein (IHC) | HPA → |
| Small intestine | Glandular cells | High | Protein (IHC) | HPA → |
| Testis | Elongated or late spermatids | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ACSL6 IHC Tips
Troubleshoot ACSL6 chromogenic IHC in paraffin sections using the selected antibody caption, expression references, and standard section controls.
How should I retrieve ACSL6 when paraffin sections stain weakly?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 minutes (page retrieval rule: cytoplasmic or membrane antigen). Let sections cool in the buffer, then compare retrieved and unretrieved serial sections with the same antibody incubation and chromogen development (standard IHC practice). The selected antibody has a paraffin-section image at 1:50, but its caption gives no retrieval conditions (selected caption: A07813-2). If signal remains weak, test a more alkaline retrieval buffer on adjacent sections as a fallback, checking tissue preservation and background before interpreting any gain (standard IHC practice).
Could fixation explain absent ACSL6 staining in my paraffin sections?
ACSL6-specific sensitivity to fixative type or fixation duration is unknown from the supplied evidence (selected caption: A07813-2; fixative not stated). Record the actual fixative and processing history, and compare sections processed together before changing antibody concentration or retrieval conditions (standard IHC practice). Excessive or uneven fixation can alter antigen accessibility in IHC generally, so examine staining across the section and repeat the citrate pH 6.0, 20-minute retrieval consistently (standard IHC practice; page retrieval rule). Do not attribute a weak result to ACSL6 fixation sensitivity solely from tissue-expression patterns or predicted membrane topology (HPA tissue IHC; UniProt Q9UKU0 topology).
What staining pattern is plausible for ACSL6 in tissue IHC?
Evaluate cytoplasmic or punctate staining rather than requiring a nuclear pattern: tissue IHC describes mainly cytoplasmic expression, while cell imaging places ACSL6 mainly in vesicles (HPA tissue IHC; HPA subcellular). UniProt also assigns ACSL6 to mitochondrial outer, peroxisomal, microsomal, and endoplasmic reticulum membranes, with residues 46–697 on the cytoplasmic side (UniProt Q9UKU0 localisation and topology). At routine light-microscope resolution, do not identify an individual organelle from brown chromogen alone (standard IHC interpretation). Compare the stained compartment with adjacent unstained cells and a no-primary control, keeping counterstain and DAB development consistent (standard IHC practice).
Can the antibody distinguish ACSL6 isoforms in paraffin sections?
Treat isoform-specific staining as unproven: the record lists 9 ACSL6 isoforms, but the supplied antibody caption does not map its epitope (UniProt Q9UKU0 isoforms; selected caption: A07813-2). The annotated transmembrane segment spans residues 25–45, followed by a cytoplasmic region at 46–697; these coordinates do not establish which region this antibody recognizes (UniProt Q9UKU0 topology). Obtain an epitope sequence or validation data before claiming coverage of any splice variant (standard antibody-validation practice). For troubleshooting, compare serial sections under identical retrieval and antibody conditions, and report signal as ACSL6 immunoreactivity without assigning an isoform (standard IHC practice).
How can IF help assess an ambiguous ACSL6 IHC pattern?
Use IF as a separate corroborating experiment, because the selected caption documents paraffin-section IHC at 1:50 and supplies no IF conditions for that antibody (selected caption: A07813-2). Multiplex ACSL6 with a marker of the cell population under study, then check whether signals occupy the same cells without treating overlap as proof of binding specificity (standard IF practice). Choose spectrally separated fluorophores after examining tissue autofluorescence, and include single-stain and no-primary controls (standard IF practice). If the epitope lies in the cytoplasmic region at residues 46–697, optimise permeabilisation to expose that membrane-facing region without dispersing puncta (UniProt Q9UKU0 topology; standard IF practice).
How do I separate diffuse ACSL6 staining from IHC background?
Compare a no-primary section with the stained section before increasing antibody concentration or DAB development time (standard IHC practice). Block endogenous peroxidase for an HRP–DAB workflow and use an appropriate protein block; these are general detection controls, not ACSL6-specific validation (standard IHC practice). The selected paraffin-section caption reports 1:50, so assess that dilution alongside a more dilute condition while holding retrieval and development constant (selected caption: A07813-2; standard IHC practice). Distrust uniform staining over damaged edges or necrotic areas, especially when the expected cellular cytoplasmic pattern disappears (HPA tissue IHC profile; standard IHC interpretation).
How should I score ACSL6 chromogenic staining across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because the tissue reference describes mainly cytoplasmic ACSL6 staining (HPA tissue IHC profile). Record the percentage of positive cells and intensity on a 0–3 scale, then calculate an H-score from 0–300 if intensity matters (standard IHC scoring practice). For sparse populations, report positive cells per mm² of viable tissue, with the denominator restricted to the same annotated region in every specimen (standard IHC quantification practice). Keep section processing, counterstain, imaging, thresholds, and observer rules constant, and normalise comparisons to eligible cells or viable area rather than whole-slide area (standard IHC practice).
Which ACSL6-positive cells and patterns should I trust?
Look for reproducible cytoplasmic staining in anatomically identified cells: HPA reports high signal in seminal-vesicle and small-intestinal glandular cells and in elongated or late spermatids (HPA tissue IHC). Use cell identity and compartment together, since HPA reports no detectable signal in breast adipocytes and bone-marrow hematopoietic cells, despite broader ACSL6 expression descriptions elsewhere (HPA tissue IHC; UniProt Q9UKU0 tissue specificity). A predominantly nuclear signal, section-edge gradient, necrotic-area deposit, or staining persisting without primary antibody warrants investigation before scoring (HPA tissue IHC profile; standard IHC interpretation). Check endogenous enzyme background with the detection controls and interpret the selected breast-carcinoma image only as a paraffin-section antibody example at 1:50 (standard IHC practice; selected caption: A07813-2).
Boster reagents
Best ACSL6 / Long-chain-fatty-acid--CoA ligase 6 IHC Antibodies
Anti-ACSL6 antibodies list IHC and IF applications across human, mouse and rat (catalog applications/reactivity); the supplied image documents IHC in a human paraffin section (catalog IHC image caption).
Real IHC data
Anti-ACSL6 (D531) Antibody
Cat # A07813-2
The rendered card is A07813-2, listed for IHC in human, mouse and rat (catalog applications/reactivity). Its own figure shows paraffin-embedded human breast carcinoma at 1:50 (catalog IHC image caption).
Which to pick: For tissue IHC, choose A07813-2: its own figure documents a human paraffin section, and the fixative is unreported (catalog IHC image caption). For IF/ICC, consider the polyclonal A07813-1 because IF is listed; ICC-specific testing and an IF image are unreported (catalog dilution raw; catalog applications; catalog image alts). Both SKUs list human, mouse and rat reactivity, but the supplied tissue image documents human tissue only (catalog reactivity; catalog IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9UKU0 (ACSL6_HUMAN, Long-chain-fatty-acid--CoA ligase 6).
- Human Protein Atlas. ACSL6 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. ACSL6 subcellular location (ICC-IF): Mainly localized to vesicles. In addition localized to the cytosol..
- Human Protein Atlas. ACSL6 antibody validation summary (1 antibodies).
- A ferroptosis associated gene signature for predicting prognosis and immune responses in patients with colorectal carcinoma. Frontiers in genetics 2022 — PMC9493326.
- ACSL6-activated IL-18R1-NF-κB promotes IL-18-mediated tumor immune evasion and tumor progression. Science advances 2024 — PMC11409972.
- Circular RNA circBNC2 inhibits tumorigenesis by modulating ferroptosis and acts as a nanotherapeutic target in prostate cancer. Molecular cancer 2025 — PMC11759416.
- PubMed PMID:10548543 — UniProt-cited evidence.
- PubMed PMID:10502316 — UniProt-cited evidence.
- PubMed PMID:16834775 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ACSL6 (Long-chain-fatty-acid--CoA ligase 6). Expected localisation Cytoplasm observed; organelle membranes annotated (HPA tissue IHC; UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A07813-2 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Seminal vesicle (Glandular cells); negative Bone marrow. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A07813-2); 2. Fixation: Image fixative and duration unreported (datasheet A07813-2); 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-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-ACSL6, 1:50-1:200 (datasheet A07813-2); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Medium consistency between staining and RNA data (HPA tissue IHC); Isoform / epitope: 9 isoforms; cytoplasmic region aa 46–697 (UniProt); Regulation: Staining regulation unreported (UniProt). Evidence: UniProt Q9UKU0, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to vesicles. In addition localized to the cytosol., Datasheet A07813-2, PMC11409972, PMC11759416. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ACSL6/immunohistochemistry-ihc-guide.