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ARL6 / ADP-ribosylation factor-like protein 6 · IHC design guide
Design Immunohistochemistry for ARL6
Plan chromogenic ARL6 IHC on paraffin sections using ciliated bronchus or fallopian tube as high-staining controls (HPA tissue IHC). This guide covers fixation, antigen retrieval, antibody dilution of 1:50–1:100 (datasheet), and assessment of cytoplasmic and ciliary staining (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ARL6 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 with cilia positivity (HPA tissue IHC) | |
| Staining pattern | Mainly cytoplasmic in most tissues, including cilia (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Bronchus+4 more · see all | |
| Negative control | Adipose tissue+3 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep paraffin-section fixation consistent (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 A05003) | |
| Caveat | Antibody staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Retina-enhanced RNA; staining effect unknown (HPA tissue RNA + IHC) | |
| Isoform / epitope | 2 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended ARL6 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet) with a published mouse tissue IHC protocol (PMC5154108).
| Sample | Paraffin-embedded human prostate tissue; fixative not specified (datasheet A05003) |
| Fixation | Image fixative and duration unreported (datasheet A05003); 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-ARL6, 1:50-1:100 (datasheet A05003) |
| 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 | ARL6-positive staining in ciliated cells (cilia axoneme) of bronchus (HPA tissue IHC: High). HPA tissue profile: Mainly cytoplasmic expression in most tissues, including cilia positivity. No signal in the no-primary control. |
💡Decision noteFor paraffin sections, start with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page antigen retrieval).
Section 2
What Is the Expected ARL6 Staining Pattern?
ARL6 localizes to the cilium membrane, axoneme and basal body, with puncta near the axoneme (UniProt Q9H0F7). In paraffin sections, expect mainly cytoplasmic staining in many tissues and conspicuous cilia staining in ciliated epithelium, including bronchus, fallopian tube and nasopharynx (HPA tissue IHC). ARL6 has no transmembrane segment (UniProt Q9H0F7 topology). HPA rates its tissue IHC evidence Approved, with medium consistency between staining and RNA expression (HPA tissue IHC).
| Cilia and adjacent cytoplasm stain in bronchial, fallopian tube or nasopharyngeal ciliated cells (HPA tissue IHC). | This fits the reported high cilia axoneme signal and mainly cytoplasmic tissue profile (HPA tissue IHC). Fine puncta near an axoneme are also compatible with ARL6 localization (UniProt Q9H0F7). Judge the pattern against recognizable ciliated cells, rather than expecting every cell in the section to look alike (general IHC practice). |
| Strong signal is confined to nuclei while identifiable cilia and cytoplasm lack signal. | That is discordant with the main tissue IHC pattern (HPA tissue IHC) and warrants a specificity check. Nucleoplasm is an uncertain additional location in ICC-IF (HPA subcellular ICC-IF), so isolated nuclear chromogen in IHC should not be accepted as the expected result on that basis. |
| Intense staining appears in adipocytes or in prostate glandular cells, especially without signal in a positive control. | Both cell types are reported as not detected in tissue IHC (HPA tissue IHC). Investigate nonspecific antibody binding or endogenous detection activity with appropriate controls (general IHC practice). A single discordant cell type does not, by itself, identify which source caused the stain. |
| Chromogen covers the section diffusely, obscuring cell borders and cilia. | The reported cell and compartment pattern cannot be scored reliably through diffuse background (HPA tissue IHC; general IHC practice). Examine a no-primary control and review blocking, washes and detection conditions before interpreting weak cellular staining (general IHC practice). |
| No staining appears in an adequately preserved bronchial ciliated epithelium control. | This conflicts with the reported high bronchial cilia signal (HPA tissue IHC). Check antibody and detection controls, section quality, and the retrieval conditions specified for the antibody (general IHC practice). HPA does not establish ARL6-specific fixation sensitivity or a universally effective retrieval condition. |
💡Expected ARL6 appearanceCall an IHC result positive when identifiable ciliated cells show cilia-associated staining with a compatible cytoplasmic pattern; bronchus, fallopian tube and nasopharynx are reported high examples (HPA tissue IHC). Diffuse section-wide chromogen or strong staining limited to a reported not-detected cell type is suspect and needs controls (HPA tissue IHC; general IHC practice).
| Anatomical compartment | UniProt places ARL6 at the cilium membrane, axoneme and basal body, including puncta flanking the axoneme (UniProt Q9H0F7). HPA describes mainly cytoplasmic tissue staining with cilia positivity (HPA tissue IHC). Read ciliary signal in its cellular context. |
| Tissue and cell selection | HPA reports high signal in bronchial, fallopian tube and nasopharyngeal ciliated cells, and in kidney tubular cells (HPA tissue IHC). Adipocytes and prostate glandular cells are reported not detected (HPA tissue IHC). Select controls by cell type as well as tissue. |
| Strength of tissue evidence | The tissue IHC reliability label is Approved, with medium consistency between antibody staining and RNA expression (HPA tissue IHC). This supports a working reference pattern, while discordant staining still requires slide-level controls. |
| Isoforms and antibody coverage | Two ARL6 isoforms are listed (UniProt Q9H0F7). Neither their tissue-specific distribution nor recognition of each isoform by the selected antibody is established in the supplied record; avoid assigning a stained compartment or a negative result to one isoform. |
| Membrane association | ARL6 has no transmembrane segment, although its reported localization includes ciliary membrane and small membrane-associated patches (UniProt Q9H0F7). A ciliary outline or puncta can fit this localization; a transmembrane staining requirement would be unsupported. |
| IF/ICC cross-check | Q: Should IF/ICC reproduce the chromogenic tissue pattern? A: Use its separate guide. HPA supports primary cilium and cilium-tip localization in ICC-IF, but marks microtubules, cytosol and nucleoplasm uncertain (HPA subcellular ICC-IF). These observations do not establish an IHC-P protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| Cilia are hard to distinguish from surrounding cytoplasmic stain. | ARL6 tissue staining is mainly cytoplasmic and includes cilia positivity (HPA tissue IHC); crowded or poorly resolved structures can complicate compartment scoring (general IHC practice). | Locate intact, recognizable ciliated epithelium and compare cilia with adjacent cytoplasm at suitable magnification (general IHC practice). Record cytoplasmic and ciliary signal separately rather than scoring a uniformly brown edge as cilia. |
| The expected positive tissue has no signal. | Possible assay or specimen failure is a general IHC consideration; HPA reports high staining in bronchial ciliated cells (HPA tissue IHC). No ARL6-specific fixation effect is documented here. | Confirm that ciliated cells are present, then check the antibody's IHC-P instructions, detection reagents and an appropriate positive control (general IHC practice). Review retrieval as an assay variable without assuming an ARL6-specific optimum. |
| A reported not-detected cell type stains strongly. | Adipocytes, prostate glandular cells, cervix glandular cells and vaginal squamous epithelial cells are reported not detected (HPA tissue IHC). Nonspecific binding or endogenous detection activity can produce misleading chromogen (general IHC practice). | Compare a no-primary control, inspect whether staining follows cell anatomy, and review blocking and detection steps (general IHC practice). Treat persistent discordance as unresolved rather than reclassifying the cell type as ARL6-positive. |
| Background is widespread, including spaces without cells. | This pattern does not match the reported cellular and ciliary localization (HPA tissue IHC). In chromogenic IHC, excess detection background or inadequate washing can obscure a specific pattern (general IHC practice). | Use a no-primary control and review wash, blocking and detection conditions (general IHC practice). Reassess ARL6 only after cell borders and cilia can be evaluated. |
| Nuclear staining dominates the section. | Mainly cytoplasmic tissue staining is reported (HPA tissue IHC). Nucleoplasm is listed only as an uncertain additional ICC-IF location (HPA subcellular ICC-IF). | Check controls and compare nuclear signal with ciliary and cytoplasmic staining in a known-positive tissue (general IHC practice; HPA tissue IHC). Do not use the uncertain ICC-IF annotation alone to validate nuclear IHC staining. |
Experiment evidence
IHC & IF images showing ARL6 staining
A05003 · IHCImmunohistochemistry of paraffin-embedded human prostate using ARL6 antibody at dilution of 1:100 .Perform microwave antigen retrieval with
A05003 · IHCImmunohistochemistry of paraffin-embedded human colon using ARL6 antibody at dilution of 1:100 .Perform microwave antigen retrieval with 10
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000113966-ARL6/tissue/cerebral+cortex#imid_5111300
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000113966-ARL6/tissue/colon#imid_5111234
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000113966-ARL6/tissue/liver#imid_5111235
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000113966-ARL6/tissue/kidney#imid_5111220
HPA · ICC-IF U2OSHuman Protein Atlas immunofluorescence, U2OS, antibody HPA019361; green = ARL6, blue = nucleus, red = microtubules.
HPA · ICC-IF U2OSHuman Protein Atlas immunofluorescence, U2OS, antibody HPA019361; green = ARL6, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ARL6 IHC & IF
🧪Run bronchus first and look for ARL6 staining in the cilia axonemes of ciliated cells (HPA: Bronchus, ciliated cells [cilia axoneme], High). Use adipose tissue as the biological negative (HPA: adipocytes, Not detected); within the bronchus slide, cells without cilia should lack the cilia-axoneme staining pattern, though other ARL6 signal may occur (UniProt Q9H0F7 subcellular localization).
Positive control tissue: Bronchus (Ciliated cells (cilia axoneme), HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ARL6 in U2OS, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), with annotated localisation: Microtubules (uncertain), Primary cilium (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host and immunoglobulin class; for a polyclonal primary, use nonimmune immunoglobulin from the same host (standard IHC practice). A confirmed ARL6 knockout specimen provides a stronger biological specificity control; quench endogenous peroxidase for chromogenic bronchus IHC and check the ciliated surface for background deposits that could mimic axonemal staining (HPA: Bronchus, ciliated cells [cilia axoneme], High; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A05003 paraffin-section caption does not state a fixative (selected SKU A05003 tissue-IHC caption). That caption uses microwave retrieval in 10 mM PBS, pH 7.2, before staining at 1:100; it demonstrates a retrieval condition, not that retrieval is required (selected SKU A05003 tissue-IHC caption). IF may help resolve ciliary localization because HPA supports primary-cilium and cilium-tip signal in ICC-IF images, but the supplied evidence does not establish that frozen sections are easier; assess apical background carefully in bronchus (HPA: subcellular ICC-IF; HPA: Bronchus, ciliated cells [cilia axoneme], High).
HPA tissue IHC evidence for ARL6
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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 |
|---|---|---|---|---|
| Bronchus | Ciliated cells (cilia axoneme) | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Fallopian tube | Ciliated cells (cilia axoneme) | High | Protein (IHC) | HPA → |
| Kidney | Cells in tubules | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Prostate | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Vagina | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ARL6 IHC Tips
Troubleshoot ARL6 staining in paraffin section IHC by checking retrieval, compartment, controls and scoring before interpreting chromogenic signal.
What retrieval conditions should I try first for ARL6 in paraffin sections?
Start ARL6 paraffin section IHC with citrate buffer at pH 6.0, 95–98 °C for 20 min (page retrieval protocol). Cool sections consistently before applying the antibody, and compare a known positive ciliated tissue across runs (standard IHC practice; HPA: high in bronchial ciliated cells). If staining remains weak, test microwave retrieval in 10 mM PBS at pH 7.2 as a fallback on matched sections (A05003 prostate IHC caption). That caption reports the fallback with antibody dilution 1:100 in paraffin embedded prostate, but does not identify a fixative (A05003 prostate IHC caption).
Could fixation explain weak ARL6 staining in my sections?
ARL6 specific sensitivity to fixation is unknown from the supplied evidence; record the actual fixative and fixation duration for each specimen (supplied evidence; standard IHC practice). The A05003 image caption identifies paraffin embedded prostate and 1:100 antibody dilution, but leaves the fixative unstated (A05003 prostate IHC caption). Compare sections with documented, consistent processing and include a positive tissue in the same staining run (standard IHC practice; HPA: high in bronchial ciliated cells). If only one processing batch loses signal, repeat staining with matched retrieval and detection conditions before assigning the difference to ARL6 expression (standard IHC practice).
Where should convincing ARL6 chromogenic staining appear?
Examine cilia and the adjacent apical region closely: ARL6 is reported at the cilium membrane, axoneme and basal body, with puncta beside the axoneme (UniProt Q9H0F7 localisation). HPA reports mainly cytoplasmic tissue staining, including cilia positivity, and high staining in bronchial and fallopian tube ciliated cells (HPA tissue IHC). Compare the suspected signal with cell morphology and a ciliary marker on an adjacent section when the structures are difficult to resolve (standard IHC practice). Diffuse cytoplasmic staining alone is less specific evidence of ciliary localisation; assess it alongside the ciliary pattern and controls (HPA tissue IHC; standard IHC practice).
Can an ARL6 epitope or isoform explain discordant staining?
ARL6 has 2 annotated isoforms, while the supplied antibody caption gives no epitope or isoform specificity (UniProt Q9H0F7 isoforms; A05003 prostate IHC caption). Check the antibody's immunogen documentation before treating a negative section as absence of both isoforms (standard IHC practice). The annotated protein has no transmembrane segment or glycosylation sites, and its listed mature chain spans residues 2–186 (UniProt Q9H0F7 topology, glycosylation and processing). Those annotations do not identify which residues this antibody recognizes or establish how fixation affects that epitope (UniProt Q9H0F7 annotations; supplied antibody evidence).
How should I assess ARL6 localisation by multiplex IF?
On the separate IF/ICC guide, pair ARL6 with a ciliary marker and a marker identifying the expected ciliated cell population, then inspect overlap at individual cilia (standard IF practice; HPA: cilia positivity). Use spectrally separated fluorophores and place the weaker signal in a red or far red channel after checking tissue autofluorescence (standard IF practice). ARL6 lacks an annotated transmembrane segment and is reported at ciliary membrane associated patches; select gentle permeabilisation according to the antibody's documented epitope accessibility (UniProt Q9H0F7 topology and localisation; standard IF practice). Include single stain controls to assess bleed through before interpreting apparent colocalisation (standard IF practice).
How do I distinguish ARL6 signal from chromogenic background?
Inspect no primary controls and adjacent tissue before increasing the catalog antibody concentration (standard IHC practice). Quench endogenous peroxidase before HRP and DAB detection, and use an appropriate protein block and thorough washes to limit nonspecific colour (standard chromogenic IHC practice). Compare background at tissue edges, folds and damaged areas with intact ciliated epithelium, where ARL6 cilia positivity is reported (standard IHC practice; HPA tissue IHC). The A05003 caption documents 1:100 in paraffin embedded prostate; that image alone does not establish the specificity of every brown deposit (A05003 prostate IHC caption; standard IHC practice).
How should I quantify ARL6 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the compartment and eligible cell population before scoring, and apply the same threshold to every section (standard IHC practice). For cellular staining, report the percentage of positive cells and an H score from 0–300, calculated as the sum of intensity category multiplied by its percentage (standard IHC scoring practice). For resolved ciliary puncta, report positive puncta density per mm² of viable sampled tissue, or positive cilia per counted ciliated cell (standard image analysis practice; UniProt Q9H0F7 localisation). Normalise comparisons to the same eligible cell type and sampled tissue area, excluding folds and necrosis by a predefined rule (standard IHC practice).
When is ARL6 staining persuasive rather than artefactual?
Give greatest weight to reproducible ciliary or adjacent apical punctate signal in morphologically appropriate cells with clean controls (UniProt Q9H0F7 localisation; standard IHC practice). HPA reports high ARL6 staining in bronchial ciliated cells but no detection in prostate glandular cells, despite the A05003 paraffin prostate image (HPA tissue IHC; A05003 prostate IHC caption). Treat that discrepancy as a reason to verify cell identity, staining controls and independent evidence before calling prostate staining ARL6 positive (standard IHC practice). Diffuse nuclear colour, isolated tissue edge staining, necrotic deposits or colour present in a no primary control warrants investigation for artefact or endogenous enzyme activity (standard IHC practice).
Boster reagents
Best ARL6 / ADP-ribosylation factor-like protein 6 IHC Antibodies
The catalog includes one anti-ARL6 antibody with real IHC images of paraffin-embedded human prostate, colon, and liver damage samples; human and mouse reactivity is listed (A05003 image captions; catalog: reactivity).
Real IHC data
Anti-ARL6 Antibody
Cat # A05003
A05003 is listed for IHC and has images of paraffin-embedded human prostate, colon, and liver damage samples at 1:100 (catalog: applications; A05003 image captions). Mouse reactivity is listed, but the supplied images show human samples only (catalog: reactivity; A05003 image captions).
Which to pick: Choose A05003 for tissue IHC: its application list includes IHC, and its images document paraffin-embedded human tissue with microwave retrieval in 10 mM PBS, pH 7.2; the fixative is unreported (catalog: applications; A05003 image captions). No IF/ICC application or image is supplied for A05003, so the payload does not support an IF/ICC choice (catalog: applications; catalog: IF images). A05003 lists human and mouse reactivity, but mouse tissue IHC is not shown; its host is rabbit and clonality is unreported (catalog: reactivity and host; A05003 image captions; catalog: clone).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9H0F7 (ARL6_HUMAN, ADP-ribosylation factor-like protein 6).
- Human Protein Atlas. ARL6 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ARL6 subcellular location (ICC-IF): Mainly localized to the microtubules and primary cilium. In addition localized to the nucleoplasm, cytosol and primary cilium tip..
- Human Protein Atlas. ARL6 antibody validation summary (1 antibodies).
- Small GTPase Arl6 controls RH30 rhabdomyosarcoma cell growth through ciliogenesis and Hedgehog signaling. Cell & bioscience 2016 — PMC5154108.
- Identification and functional analysis of the vision-specific BBS3 (ARL6) long isoform. PLoS genetics 2010 — PMC2841623.
- PubMed PMID:11230166 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16641997 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ARL6 (ADP-ribosylation factor-like protein 6). Expected localisation Cytoplasmic staining with cilia positivity (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A05003 at 1:50-1:100 (the product's own IHC validation image is shown). Controls (HPA): positive Bronchus (Ciliated cells (cilia axoneme)); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human prostate tissue; fixative not specified (datasheet A05003); 2. Fixation: Image fixative and duration unreported (datasheet A05003); 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-ARL6, 1:50-1:100 (datasheet A05003); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep paraffin-section fixation consistent (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 A05003); Caveat: Antibody staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage is unspecified (UniProt); Regulation: Retina-enhanced RNA; staining effect unknown (HPA tissue RNA + IHC). Evidence: UniProt Q9H0F7, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the microtubules and primary cilium. In addition…, Datasheet A05003, PMC5154108. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ARL6/immunohistochemistry-ihc-guide.