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ACSL6 / Long-chain-fatty-acid--CoA ligase 6 · IHC design guide
Design Immunohistochemistry for ACSL6
Plan ACSL6 staining in paraffin sections using its observed cytoplasmic tissue pattern and documented membrane topology (HPA tissue IHC; UniProt). The guide highlights positive tissue choices, fixation consistency and interpretation of the bone marrow discrepancy (HPA tissue IHC; UniProt).
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 | Cytoplasmic staining; membrane association is molecular (HPA tissue IHC; UniProt) | |
| Staining pattern | Cytoplasmic in glandular cells, spermatids and brain cells (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 sections (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A07813-2) | |
| Caveat | Bone marrow IHC is undetected despite precursor expression (HPA tissue IHC; UniProt) | |
| Regulation | Staining intensity varies by tissue and cell type (HPA tissue IHC) | |
| Isoform / epitope | 9 isoforms; check epitope coverage of the cytoplasmic region (UniProt) |
Section 1
Recommended ACSL6 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with the published ACSL6 paraffin-section protocol (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 HIER at 95–98 °C for 20 min (page retrieval rule); the published protocol does not specify retrieval (PMC11759416).
Section 2
What Is the Expected ACSL6 Staining Pattern?
ACSL6 should appear predominantly cytoplasmic in glandular cells of seminal vesicle and small intestine and in elongated or late spermatids in testis (HPA tissue IHC: High). UniProt places ACSL6 on mitochondrial outer, peroxisomal, microsomal and endoplasmic reticulum membranes, with residues 46–697 facing the cytoplasm (UniProt Q9UKU0 topology). HPA rates its tissue staining Enhanced, with medium consistency against RNA expression and external verification pending (HPA tissue IHC).
| Cytoplasmic staining in seminal vesicle or small intestine glandular cells, or in elongated or late testis spermatids. | These are the clearest reported positive patterns: each cell population is scored High by HPA (HPA tissue IHC). Assess the named cells, since a positive area alone does not establish that the expected population stained. |
| Predominantly nuclear staining, especially when cytoplasmic staining is absent. | This conflicts with the reported cytoplasmic tissue pattern and membrane associated topology (HPA tissue IHC; UniProt Q9UKU0 topology). Treat it as suspect and review staining controls; the supplied sources do not establish nuclear ACSL6 as an expected IHC result. |
| Strong staining in a cell population reported as undetected, such as epididymis glandular cells. | HPA reports no detection in those cells (HPA tissue IHC). Check for cross-reactivity or endogenous detection activity before calling the result ACSL6 positive. An HPA negative is a comparison point, not proof that every specimen must be negative. |
| Diffuse color across tissue and surrounding areas, without a clear cellular pattern. | This is difficult to score as specific cytoplasmic staining (HPA tissue IHC: cytoplasmic profile). Compare a no-primary control and inspect blocking, washing and chromogen development as general IHC practice; the pattern alone cannot identify its cause. |
| No signal in the expected cells of a seminal vesicle, small intestine or testis section. | HPA reports High staining in the specified populations (HPA tissue IHC). First confirm those cells are present, then review the antibody's validated IHC-P workflow and detection controls. A negative section alone cannot distinguish technical failure from specimen variation. |
💡Expected ACSL6 appearanceCall a section positive when the named glandular cells or elongated or late spermatids show clear cytoplasmic staining at the reported High level; isolated nuclear staining or color outside the expected cells warrants control review (HPA tissue IHC; UniProt Q9UKU0 topology).
| Cell population and tissue selection | HPA scores seminal vesicle and small intestine glandular cells and testis elongated or late spermatids High; cerebellar Purkinje cells and cerebral cortex glial cells are Medium (HPA tissue IHC). Record the population examined when comparing sections. |
| Membrane topology | ACSL6 has one transmembrane segment at residues 25–45, with residues 46–697 cytoplasmic (UniProt Q9UKU0 topology). This supports a cytoplasmic compartment interpretation, but no antibody epitope is supplied, so topology cannot predict retrieval needs. |
| Alternative isoforms and processing | UniProt lists 9 isoforms and a single chain spanning residues 1–697, with no signal peptide or propeptide annotated (UniProt Q9UKU0). Without an antibody epitope, the supplied record cannot establish which isoforms the stain detects. |
| IHC evidence and RNA comparison | HPA calls the tissue IHC pattern Enhanced, describes medium consistency with RNA expression, and notes that external verification is pending (HPA tissue IHC). Tissue enhanced RNA expression in bone marrow does not override HPA's undetected bone marrow hematopoietic cell staining. |
| Q: What should IF/ICC show? | A: HPA reports mainly vesicles, with additional cytosol localization marked uncertain; its listed antibody HPA040470 has ICC status Uncertain (HPA subcellular; HPA antibodies). Interpret that evidence on the separate IF/ICC guide page, without treating its images as an IHC-P protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive cells show no chromogenic signal. | The cell population may be absent from the section, or an IHC workflow step may have failed; HPA reports those named populations as High (HPA tissue IHC). | Confirm cell identity on the counterstain and inspect positive and detection controls. Review the catalog antibody's IHC-P instructions for retrieval, primary incubation and detection as general IHC practice. |
| Only nuclei stain strongly. | A nuclear dominant result differs from HPA's cytoplasmic tissue profile and UniProt's membrane associated topology (HPA tissue IHC; UniProt Q9UKU0). | Compare the expected cytoplasmic compartment in a reported positive tissue and check a no-primary control. Do not score nuclear color alone as the expected ACSL6 pattern. |
| An HPA negative cell population stains strongly. | Cross-reactivity or endogenous detection activity is possible; HPA reports epididymis glandular cells and bone marrow hematopoietic cells as Not detected (HPA tissue IHC). | Compare the questionable cells with a reported positive population and a no-primary control. If signal persists without primary antibody, review the detection blocking steps as general IHC practice. |
| Background color obscures cellular boundaries. | The result cannot be assigned confidently to HPA's cytoplasmic pattern (HPA tissue IHC); background may arise during detection or chromogen development (general IHC practice). | Inspect the no-primary control, then review blocking, washes and development time under the applicable IHC-P workflow. Reassess only where individual cells remain interpretable. |
| Bone marrow IHC is negative despite expected expression from other evidence. | UniProt describes expression in erythrocyte precursors and hematopoietic sources, while HPA reports bone marrow hematopoietic cells as Not detected by tissue IHC (UniProt Q9UKU0 tissue specificity; HPA tissue IHC). | Report the IHC observation with its cell population. Use an HPA High tissue pattern as the staining comparison; do not infer that all marrow cell populations share one protein staining level. |
| Vesicular IF/ICC images seem different from diffuse cytoplasmic IHC. | HPA reports mainly vesicular ICC-IF localization, while its tissue IHC profile is cytoplasmic; HPA040470 is ICC Uncertain (HPA subcellular; HPA tissue IHC; HPA antibodies). | Judge the paraffin section against the tissue IHC cell and compartment pattern. Consult the separate IF/ICC guide for image interpretation; do not use ICC-IF localization to set IHC-P conditions. |
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 seminal vesicle first and look for ACSL6 staining in its glandular cells (HPA: High in seminal-vesicle glandular cells). Use epididymis glandular cells as the negative tissue (HPA: Not detected in epididymis glandular cells); compare nonglandular cells on the seminal-vesicle slide with background, 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 no-primary (secondary-only) and host-species-matched nonimmune IgG controls for the polyclonal primary (caption: pAb), plus ACSL6-knockout material if available or a peptide-block control if the immunizing peptide is available. For chromogenic IHC, block endogenous peroxidase and check whether luminal material in seminal vesicle retains nonspecific signal (standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported; the A07813-2 paraffin-section caption specifies 1:50 in breast carcinoma but leaves the fixative unreported (selected IHC caption). Antigen-retrieval dependency is unreported, so optimize retrieval against the positive and negative tissues; available ICC-IF images do not establish that IF or frozen sections are easier (HPA subcellular: U2OS and KOLF2.1J images). In seminal vesicle, distinguish glandular-cell staining from signal in luminal secretions when scoring (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 staining in paraffin sections by checking retrieval, cellular pattern, controls, and scoring before interpreting chromogenic signal.
How should I retrieve ACSL6 in paraffin sections when staining is weak?
Start ACSL6 paraffin IHC with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval setting: cytoplasmic or membrane antigen). Let sections cool in the retrieval buffer, then process a reference tissue alongside the test sections so staining changes can be compared under the same detection conditions (standard IHC practice). If signal remains weak, vary heating time in small increments before testing a different buffer or pH as a fallback (standard IHC optimisation). Check whether background rises as signal improves, and retain the retrieval condition that preserves tissue morphology and interpretable cytoplasmic staining (standard IHC practice; HPA tissue IHC: cytoplasmic profile).
Could fixation explain inconsistent ACSL6 staining across paraffin blocks?
ACSL6-specific sensitivity to fixation is unknown from the supplied evidence; the breast carcinoma caption reports paraffin embedding but does not state a fixative (A07813-2 tissue-IHC caption). For new specimens, record the fixative, fixation duration, and processing conditions for each block, then compare sections processed together (standard IHC practice). If archival blocks differ, use matched reference sections and hold retrieval, antibody incubation, and chromogenic development constant while investigating the variation (standard IHC practice). Avoid attributing weak signal to a particular fixation effect without a controlled comparison, and assess tissue preservation before scoring cells (standard IHC practice).
What cellular pattern should ACSL6 show in chromogenic IHC?
Assess ACSL6 primarily as cytoplasmic staining in tissue sections, while recording whether the signal is diffuse or punctate (HPA tissue IHC: cytoplasmic expression; HPA subcellular: vesicles and cytosol). Its annotated sites include mitochondrial outer, peroxisomal, microsomal, and endoplasmic reticulum membranes, with residues 46–697 on the cytoplasmic side (UniProt Q9UKU0 localisation and topology). Chromogenic IHC may not resolve those organelles individually, so do not assign a brown punctum to a specific membrane without an independent marker (standard IHC interpretation). Compare the pattern with cell morphology and a negative control; predominantly nuclear staining warrants investigation before it is scored as ACSL6 (standard IHC practice; UniProt Q9UKU0 localisation).
How can ACSL6 isoforms or an unknown epitope complicate IHC interpretation?
ACSL6 has 9 listed isoforms, so staining across variants depends on which sequence the antibody recognises (UniProt Q9UKU0 isoforms; standard antibody interpretation). The supplied caption establishes staining with A07813-2 in paraffin-embedded breast carcinoma at 1:50, but does not identify its epitope or establish recognition of every isoform (A07813-2 tissue-IHC caption). Compare the antibody’s documented immunogen or epitope, if available, with sequences of the isoforms relevant to the specimen before drawing isoform-specific conclusions (standard antibody validation practice). Because the annotated transmembrane segment spans residues 25–45 and the remaining 46–697 are cytoplasmic, epitope accessibility also depends on the actual recognised sequence and processing conditions (UniProt Q9UKU0 topology; standard IHC practice).
How should I use IF to investigate an ambiguous ACSL6 IHC pattern?
Use IF as a complementary localisation check rather than treating the chromogenic paraffin caption as IF validation (A07813-2 tissue-IHC caption; standard assay validation practice). Multiplex ACSL6 with a validated marker for the expected cell type, such as a Purkinje-cell marker when examining cerebellum, and include single-stain controls to check channel bleed-through (HPA tissue IHC: medium staining in Purkinje cells; standard IF practice). Choose fluorophores after examining tissue autofluorescence in unstained sections, favouring channels with clearer separation from that background (standard IF practice). If the antibody recognises an epitope within the cytoplasmic residues 46–697, optimise permeabilisation to reach that side of the membrane while preserving morphology; the supplied evidence does not locate this antibody’s epitope (UniProt Q9UKU0 topology; standard IF practice).
What should I check when ACSL6 chromogenic staining is widespread or diffuse?
First inspect a section processed without primary antibody to identify signal from the detection system, then compare it with the complete stain (standard IHC practice). Use an appropriate endogenous peroxidase block before an HRP and DAB workflow, and evaluate nonspecific binding with suitable protein blocking and washing (standard chromogenic IHC practice). Titrate the primary antibody around the caption’s 1:50 condition only as an optimisation exercise; that caption documents a breast carcinoma image, not a universal dilution (A07813-2 tissue-IHC caption). Diffuse cytoplasmic staining can be plausible, but uniform staining across unrelated structures or tissue edges should prompt review of controls, development time, and morphology (HPA tissue IHC: cytoplasmic profile; standard IHC practice).
How should I score ACSL6 staining across different tissue compartments? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, because ACSL6 staining varies among reported cell types (HPA tissue IHC: high in seminal-vesicle glandular cells and late spermatids; medium in Purkinje cells). For chromogenic sections, report the percentage of positive cells and an intensity-based H-score, or positive-cell density per mm² when cell counts are the relevant endpoint (standard IHC quantification practice). Normalise counts to the corresponding viable cell population or measured tissue area, and exclude folds, edges, and necrotic regions using preset rules (standard IHC practice). Apply one staining threshold and imaging or scoring procedure across comparison groups, while documenting the antibody, retrieval, and detection conditions (standard IHC practice).
How can I distinguish credible ACSL6 staining from artefact?
A credible result has cytoplasmic signal in an anatomically identified cell population, with appropriate control behaviour and preserved morphology (HPA tissue IHC: cytoplasmic profile; standard IHC interpretation). For example, high staining is reported in seminal-vesicle glandular cells and elongated or late spermatids, while breast adipocytes are reported as not detected (HPA tissue IHC). Investigate isolated nuclear signal, sharp edge-only staining, necrotic debris, or signal persisting without primary antibody before calling a specimen positive (UniProt Q9UKU0 membrane localisation; standard IHC practice). Check the peroxidase block and detection controls if brown precipitate appears independently of the primary antibody (standard chromogenic IHC practice). Treat an unexpected pattern cautiously because the HPA tissue-IHC assessment is Enhanced with medium staining–RNA consistency and pending external verification (HPA tissue IHC reliability).
Boster reagents
Best ACSL6 / Long-chain-fatty-acid--CoA ligase 6 IHC Antibodies
An ACSL6 IHC image shows a paraffin-embedded human specimen (A07813-2 image caption). The catalog lists human, mouse, and rat reactivity for both antibodies and IF for A07813-1 (catalog: applications/reactivity).
Real IHC data
Anti-ACSL6 (D531) Antibody
Cat # A07813-2
The rendered card is A07813-2, with IHC shown in paraffin-embedded human breast carcinoma at 1:50 (A07813-2 image caption). Human, mouse, and rat IHC reactivity is listed, while the supplied image documents a human specimen (catalog: A07813-2 applications/reactivity; A07813-2 image caption).
Which to pick: For tissue IHC, choose A07813-2: its image documents paraffin-embedded human breast carcinoma at 1:50, but does not report the fixative (A07813-2 image caption). For IF, consider A07813-1, a polyclonal antibody listed for IF at 1:50; no IF image or ICC validation is supplied (catalog: A07813-1 dilution_raw/applications/if_image_alts). For cross-species IHC, both list human, mouse, and rat reactivity, although the supplied IHC image for A07813-2 shows only human tissue (catalog: applications/reactivity; A07813-2 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 Cytoplasmic staining; membrane association is molecular (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 sections (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A07813-2); Caveat: Bone marrow IHC is undetected despite precursor expression (HPA tissue IHC; UniProt); Isoform / epitope: 9 isoforms ; check epitope coverage of the cytoplasmic region (UniProt); Regulation: Staining intensity varies by tissue and cell type (HPA tissue IHC). Evidence: UniProt Q9UKU0, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to vesicles. In addition localized to the cytosol., Datasheet A07813-2, PMC11759416. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ACSL6/immunohistochemistry-ihc-guide.