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ARF6 / ADP-ribosylation factor 6 · IHC design guide
Design Immunohistochemistry for ARF6
Plan ARF6 staining in paraffin sections using its cytoplasmic and membranous tissue pattern (HPA tissue IHC). This guide covers fixation, the IHC-validated antibody, chromogenic detection, controls, and interpretation of staining that may diverge from RNA expression (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ARF6 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic and membranous tissue staining (HPA tissue IHC) | |
| Staining pattern | Most tissues show cytoplasmic and membranous staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PB9987) | |
| Positive control | Adrenal gland+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) | |
| Caveat | Antibody staining has low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | No specific expression regulator is reported (UniProt) | |
| Isoform / epitope | No annotated isoforms; one chain spans residues 2–175 (UniProt) |
Section 1
Recommended ARF6 IHC & IF Protocols
The catalog antibody uses EDTA pH 8.0 retrieval (datasheet PB9987). The published options below report ARF6 IHC in prostate, breast, zebrafish, and mouse samples (PMC4499334; PMC4336811; PMC10054615).
| Sample | Paraffin-embedded human melanoma tissue; fixative not specified (datasheet PB9987) |
| Fixation | Image fixative and duration unreported (datasheet PB9987); 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 PB9987); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9987) |
| Primary antibody | Rabbit anti-ARF6, 0.5-1μg/ml (datasheet PB9987) |
| Primary incubation | Overnight at 4 °C (datasheet PB9987) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9987) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ARF6-positive staining in glandular cells of adrenal gland (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic and membranous expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 for the catalog antibody (datasheet PB9987); use each published retrieval condition with its corresponding antibody and sample (PMC4499334; PMC10054615; PMC4336811).
Section 2
What Is the Expected ARF6 Staining Pattern?
ARF6 is a cytosolic protein that also associates with the plasma membrane and endosomal membranes; it has no transmembrane segment (UniProt P62330 topology and subcellular location). In paraffin sections, expect cytoplasmic and membranous staining in many cell types, including medium staining in several glandular epithelia (HPA tissue IHC). HPA rates the tissue IHC antibody Approved, while reporting low consistency between staining and RNA expression (HPA tissue IHC).
| Cytoplasmic staining with some cell-edge staining in glandular cells. | This fits ARF6 localization (UniProt P62330 subcellular location). HPA reports medium staining in glandular cells of colon, breast, appendix, and several other tissues (HPA tissue IHC). Judge the pattern against the cells and tissue on the slide; a uniform membrane outline is not required by either source. |
| Strong nuclear-only staining with little cytoplasmic or membrane signal. | Treat a nuclear-only pattern as suspect: the supplied localization records place ARF6 in cytosol and membrane-associated compartments, with no nuclear location listed (UniProt P62330 subcellular location; HPA ICC-IF). Check whether the signal follows counterstained nuclei and persists in the negative control before calling it ARF6. |
| Prominent staining in a cell population listed as not detected by HPA. | For example, HPA reports no detection in bone-marrow hematopoietic cells and parathyroid glandular cells (HPA tissue IHC). Unexpected staining warrants checks for antibody cross-reactivity or endogenous detection activity (general IHC practice). HPA's Approved rating has low staining-to-RNA consistency, so its negative calls are comparators, not absolute exclusions (HPA tissue IHC). |
| Diffuse color across cells, stroma, and empty slide areas. | A field-wide deposit does not match the reported cytoplasmic and membranous tissue pattern (HPA tissue IHC). Review the no-primary control, blocking, wash stringency, and detection chemistry for nonspecific background (general IHC practice). Do not score a uniformly colored field as widespread ARF6 expression. |
| No staining in glandular cells expected to be positive. | HPA reports medium staining in glandular cells of colon, breast, and appendix (HPA tissue IHC). If an appropriate reference section also lacks signal, check section quality, antibody and detection controls, and the retrieval conditions used in the IHC workflow (general IHC practice). A blank test section alone cannot establish ARF6 absence. |
💡Expected ARF6 appearanceCall a result positive when glandular cells show discernible cytoplasmic staining, possibly with membrane accentuation, around the medium level reported for the chosen HPA reference tissue; nuclear-only color or diffuse field-wide deposit is suspect (HPA tissue IHC; UniProt P62330 subcellular location).
| Compartment and topology | ARF6 occupies cytosol and several membrane-associated sites but has no transmembrane segment (UniProt P62330 topology and subcellular location). Cytoplasmic signal can therefore be compatible with membrane trafficking; membrane-only staining is not a required acceptance rule. |
| Membrane association | Myristoylation is required for proper ARF6 membrane localization (UniProt P62330 subcellular location). This supports accepting a mixed cytoplasmic and membrane pattern. It does not establish how formalin fixation or antigen retrieval will affect this assay. |
| Tissue-level confidence | HPA describes cytoplasmic and membranous expression in most tissues, yet flags low consistency between antibody staining and RNA expression (HPA tissue IHC). Use its cell-specific examples as reference patterns and record departures rather than treating every HPA negative cell as a definitive biological negative. |
| Antibody evidence | The mouse monoclonal CAB002778 has Approved IHC status; CAB079914 has no IHC status listed in the supplied antibody record (HPA antibodies). Interpret a result with the validation status of the antibody actually used. The supplied evidence gives no target-specific fixation sensitivity or retrieval condition. |
| IF/ICC: should the pattern match? | In ICC-IF, HPA supports cytosolic localization and lists A-431, U-251MG, and U2OS images (HPA ICC-IF). Use that as a compartment comparison only: the paraffin-section IHC tissue pattern includes membranous staining (HPA tissue IHC). Consult the separate IF/ICC guide for that application. |
| Situation | Likely cause | Next action |
|---|---|---|
| The expected glandular reference section is blank. | A failed primary or detection step, unsuitable section, or retrieval setting can leave an IHC assay blank (general IHC practice). | Repeat with a documented positive control, verify reagent sequence and detection, and optimize retrieval within the established IHC workflow (general IHC practice). HPA lists medium glandular staining in colon and breast as reference observations (HPA tissue IHC). |
| Nuclei dominate the signal. | A nuclear-only result conflicts with the supplied ARF6 compartments (UniProt P62330 subcellular location; HPA ICC-IF). | Compare the chromogen with the nuclear counterstain and a no-primary control; reassess antibody specificity before scoring (general IHC practice). |
| The whole section looks stained. | Nonspecific binding or detection background can obscure cell-level localization (general IHC practice). | Check a no-primary control; review blocking, washing, detection development, and section handling (general IHC practice). Score only separable cellular signal. |
| Unexpected cells stain strongly. | Cross-reactivity or endogenous detection activity is possible in IHC (general IHC practice). HPA lists some cell populations as not detected (HPA tissue IHC). | Confirm the cell identity, run appropriate detection controls, and compare the compartment with the ARF6 pattern (UniProt P62330 subcellular location). Treat HPA negatives cautiously because staining and RNA show low consistency (HPA tissue IHC). |
| Staining is cytoplasmic but lacks a crisp membrane rim. | A membrane rim need not be prominent: HPA reports cytoplasmic and membranous tissue staining, while ICC-IF supports cytosolic localization (HPA tissue IHC; HPA ICC-IF). | Assess signal in the expected cells against a positive reference section and negative controls (general IHC practice). Do not reject a controlled cytoplasmic pattern solely for lacking a sharp rim. |
| Heart muscle is negative despite the UniProt tissue note. | UniProt reports higher ARF6 levels in heart, while HPA reports cardiomyocytes as not detected by IHC (UniProt P62330 tissue specificity; HPA tissue IHC). | Record the discrepancy and rely on a demonstrated positive glandular reference for the staining run (HPA tissue IHC). Do not infer a fixation effect or assay failure from the heart result alone. |
Experiment evidence
IHC & IF images showing ARF6 staining
PB9987 · IHCIHC analysis of ARF6 using anti-ARF6 antibody (PB9987). ARF6 was detected in paraffin-embedded section of human melanoma tissue. Heat mediat
PB9987 · IHCIHC analysis of ARF6 using anti-ARF6 antibody (PB9987). ARF6 was detected in paraffin-embedded section of rat cardiac muscle tissue. Heat me
PB9987 · IFIF analysis of ARF6 using anti-ARF6 antibody (PB9987). ARF6 was detected in immunocytochemical section of A431 cells. Enzyme antigen retriev
A00927 · IHCImmunohistochemistry of ARF6 in human kidney tissue with ARF6 antibody at 5 μg/mL.
A00927 · IFImmunofluorescence of ARF6 in human kidney tissue with ARF6 antibody at 20 μg/mL.
A00927-2 · IFIF analysis of ARF6 using anti-ARF6 antibody (A00927-2). ARF6 was detected in immunocytochemical section of A431 cells. Enzyme antigen retri
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000165527-ARF6/tissue/cerebral+cortex#imid_871968
HPA · StomachHuman Protein Atlas IHC, Stomach. Source: https://www.proteinatlas.org/ENSG00000165527-ARF6/tissue/stomach+1#imid_871884
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000165527-ARF6/tissue/colon#imid_871894
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000165527-ARF6/tissue/liver#imid_871903
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB002778; green = ARF6, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB002778; green = ARF6, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ARF6 IHC & IF
🧪Run colon first: glandular cells should stain (HPA: Medium in colon glandular cells). Use bone marrow hematopoietic cells as the negative tissue (HPA: Not detected); cells without brown DAB signal on the colon slide can show local background, but no internal cell type is established as ARF6-negative (UniProt P62330: ubiquitous).
Positive control tissue: Adrenal gland (Glandular cells, HPA Medium)
Negative control tissue: Bone marrow (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ARF6 in A-431, U-251MG, U2OS, with annotated localisation: Cytosol (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, host- and isotype-matched rabbit IgG control, and ARF6 knockout material as a biological negative (selected PB9987 caption: rabbit primary). Block endogenous peroxidase and check for endogenous biotin background in colon, especially with the caption’s biotin-based DAB detection (selected PB9987 caption: SABC/DAB).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected PB9987 paraffin-section caption does not state the fixative. That caption uses heat retrieval in EDTA at pH 8.0, but a retrieval requirement is unreported; HPA documents cytosolic ICC-IF signal, while the supplied evidence does not establish whether IF or frozen sections are easier (selected PB9987 caption; HPA subcellular: cytosol supported). Check colon staining against the biotin and peroxidase controls because the reported detection method uses SABC/DAB (selected PB9987 caption).
HPA tissue IHC evidence for ARF6
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
| Colon | Glandular cells | Medium | Protein (IHC) | HPA → |
| Duodenum | Glandular 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 → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ARF6 IHC Tips
Troubleshoot ARF6 staining in paraffin sections by checking retrieval, compartment pattern and cell-specific controls before interpreting chromogenic signal.
What retrieval should I try first for weak ARF6 staining in paraffin sections?
Use heat-mediated antigen retrieval in EDTA pH 8.0 first (datasheet PB9987). The selected paraffin-section image used that retrieval before 1 µg/mL primary antibody overnight at 4°C, so those conditions provide a documented starting point for troubleshooting weak staining (datasheet PB9987). If signal remains weak, compare a modest change in heating time on adjacent sections while keeping antibody concentration and detection constant (standard IHC practice). Check morphology alongside staining, because excessive heating can damage sections and make an apparent gain in signal difficult to interpret (standard IHC practice). Record retrieval time and temperature for every comparison so a repeatable improvement can be distinguished from run variation (standard IHC practice).
Could fixation explain inconsistent ARF6 staining between paraffin blocks?
Target-specific sensitivity of ARF6 to fixation is unknown from the supplied evidence; the selected paraffin-section caption does not state a fixative (datasheet PB9987). Compare blocks only after recording their available fixation and processing histories, then stain sections together with the same EDTA pH 8.0 retrieval and detection conditions (datasheet PB9987; standard IHC practice). Include a previously staining section in each run to help separate a block effect from variation in the staining run (standard IHC practice). If morphology is damaged or staining varies across a section, assess tissue processing and section adhesion before changing the primary antibody concentration (standard IHC practice). Do not assign a particular fixation sensitivity to ARF6 without a controlled comparison.
Should ARF6 staining be cytoplasmic, membranous or punctate?
Evaluate cytoplasmic and membranous staining in paraffin sections: that is the reported pattern across most tissues (HPA tissue IHC). ARF6 is also assigned to endosomal and recycling-endosomal membranes, so fine intracellular puncta can fit its trafficking locations when morphology and controls support the signal (UniProt P62330 subcellular location). It has no annotated transmembrane segment, while myristoylation supports membrane localisation; a membrane-associated pattern does not require the protein to span the membrane (UniProt P62330 topology and subcellular location). Score the cytoplasmic, membranous and punctate components separately if they differ between cell populations (standard IHC practice). Treat isolated nuclear staining cautiously because a nuclear location is not listed in the supplied ARF6 record (UniProt P62330 subcellular location).
How should I troubleshoot a disputed ARF6 epitope or apparent isoform-specific stain?
The supplied ARF6 record lists 0 isoforms and no annotated domains, so an isoform-specific explanation for different paraffin-section patterns is unsupported here (UniProt P62330 isoforms and domains). Its epitope location is not supplied; confirm the antibody's immunogen or epitope information before attributing weak staining to masking of a particular region (datasheet PB9987, epitope not provided). ARF6 has no annotated transmembrane segment, but its membrane association makes both cellular distribution and epitope accessibility relevant to section staining (UniProt P62330 topology and subcellular location; standard IHC practice). Compare retrieval conditions on adjacent sections with the same antibody and detection reagents, and examine whether any gained signal retains the expected cytoplasmic or membranous pattern (HPA tissue IHC; standard IHC practice).
How can IF help assess an ambiguous ARF6 IHC pattern?
Use IF as a separate localisation check when chromogenic ARF6 staining cannot resolve a membrane rim from intracellular puncta (UniProt P62330 subcellular location; standard IF practice). Multiplex ARF6 with a marker for the cell population being evaluated, then compare overlap within individual cells rather than treating nearby signal as colocalisation (standard IF practice). Choose a spectrally separated, preferably far-red fluorophore for the dimmer target and inspect an unstained section for tissue autofluorescence before assigning puncta (standard IF practice). Because the antibody epitope's position is not supplied, optimise permeabilisation conditionally: test mild detergent if access to a cytosolic-facing or intracellular epitope is needed, while checking preservation of membrane detail (datasheet PB9987, epitope not provided; standard IF practice). Keep any IF result distinct from the paraffin-section IHC evidence (datasheet PB9987).
What should I check when ARF6 DAB staining is diffuse or widespread?
First compare the stained section with a no-primary control and inspect whether diffuse DAB follows tissue edges, folds or damaged regions (standard IHC practice). The documented paraffin-section workflow used 10% goat serum blocking, a biotinylated secondary antibody and streptavidin-biotin detection with DAB; each reagent step offers a place to investigate nonspecific signal (datasheet PB9987). Check peroxidase blocking and, for the biotin-based workflow, evaluate endogenous biotin as possible background sources (standard IHC practice). Titrate primary antibody around the documented 1 µg/mL condition while holding retrieval and development time fixed (datasheet PB9987; standard IHC practice). Widespread staining alone is not proof of background because HPA reports cytoplasmic and membranous expression in most tissues (HPA tissue IHC).
How should I quantify ARF6 across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, since reported ARF6 tissue staining includes both cytoplasmic and membranous expression (HPA tissue IHC). For that population, report the percentage of positive cells and an H-score from 0–300, calculated as the percentages at intensity 1, 2 and 3 multiplied by their respective intensities (standard IHC practice). If counting discrete positive cells instead, express density per mm² of viable tissue and state the analysed area (standard IHC practice). Normalise comparisons to the same cell type, viable tissue area, staining batch and scoring thresholds, and report compartments separately when their patterns differ (standard IHC practice). Exclude folds, necrosis and section edges from the scored region (standard IHC practice).
When is an ARF6-positive IHC result convincing rather than artefactual?
A convincing result shows a reproducible cellular pattern compatible with cytoplasmic or membranous ARF6, rather than signal confined to section edges or damaged tissue (HPA tissue IHC; standard IHC practice). Compare the stained cell type with a documented reference: HPA reports medium staining in glandular cells of several tissues, including colon, while its bone-marrow hematopoietic cells are listed as not detected (HPA tissue IHC). Check no-primary controls and peroxidase blocking when DAB appears in unexpected compartments or necrotic areas (standard IHC practice). Interpret reference tissues cautiously because HPA rates the tissue staining as Approved while reporting low consistency with RNA expression (HPA tissue IHC reliability). Require a repeatable, cell-resolved pattern before interpreting a difference as ARF6 biology (standard IHC practice).
Boster reagents
Best ARF6 / ADP-ribosylation factor 6 IHC Antibodies
Anti-ARF6 antibodies have IHC images from human kidney and melanoma tissue and rat cardiac muscle, plus IF images from human kidney tissue and A431 cells (catalog image captions).
Real IHC data
Anti-ARF6 Antibody ®
Cat # PB9987
Real IHC data
Anti-ADP-ribosylation factor 6 ARF6 Antibody
Cat # A00927
Real IF data
Anti-ARF6 Antibody ®
Cat # A00927-2
PB9987 has IHC images from human melanoma paraffin sections and rat cardiac muscle paraffin sections (PB9987 image captions). A00927 has human kidney IHC and IF images, while A00927-2 has an A431 cell IF image and lists ICC as an application (A00927 and A00927-2 catalog entries).
Which to pick: For paraffin-section IHC, choose PB9987: its human melanoma and rat cardiac muscle captions document EDTA retrieval at pH 8.0 and 1 μg/mL primary antibody; the fixative is unreported (PB9987 IHC image captions). A00927 is another tissue IHC option with a human kidney image at 5 μg/mL, but its caption does not report tissue processing or fixative (A00927 IHC image caption). For IF/ICC, A00927-2 has an A431 cell IF image; all three list human, mouse and rat reactivity, while the supplied IHC images document human tissue for A00927 and human and rat tissue for PB9987 (catalog applications, reactivity and image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P62330 (ARF6_HUMAN, ADP-ribosylation factor 6).
- Human Protein Atlas. ARF6 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ARF6 subcellular location (ICC-IF): Localized to the cytosol..
- Human Protein Atlas. ARF6 antibody validation summary (2 antibodies).
- Increased expression of ARF GTPases in prostate cancer tissue. SpringerPlus 2015 — PMC4499334.
- VGLL2-NCOA2 leverages developmental programs for pediatric sarcomagenesis. Cell reports 2023 — PMC10054615.
- ARF6 mediates nephrin tyrosine phosphorylation-induced podocyte cellular dynamics. PloS one 2017 — PMC5589247.
- lincRNA-RoR and miR-145 regulate invasion in triple-negative breast cancer via targeting ARF6. Molecular cancer research : MCR 2015 — PMC4336811.
- PubMed PMID:1993656 — UniProt-cited evidence.
- PubMed PMID:14659046 — UniProt-cited evidence.
- PubMed PMID:9653160 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ARF6 (ADP-ribosylation factor 6). Expected localisation Cytoplasmic and membranous tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PB9987). Validated IHC antibody: PB9987 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Bone marrow. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human melanoma tissue; fixative not specified (datasheet PB9987); 2. Fixation: Image fixative and duration unreported (datasheet PB9987); 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 PB9987); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9987); 8. Primary antibody: Rabbit anti-ARF6, 0.5-1μg/ml (datasheet PB9987); 9. Primary incubation: Overnight at 4 °C (datasheet PB9987). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Antibody staining has low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: No annotated isoforms; one chain spans residues 2–175 (UniProt); Regulation: No specific expression regulator is reported (UniProt). Evidence: UniProt P62330, HPA tissue IHC · Approved, HPA ICC-IF · cytosol., Datasheet PB9987, PMC4499334, PMC10054615. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ARF6/immunohistochemistry-ihc-guide.