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AFAP1 / Actin filament-associated protein 1 · IHC design guide
Design Immunohistochemistry for AFAP1
Plan chromogenic AFAP1 IHC on paraffin sections with the catalog antibody's 2–5 μg/mL range (datasheet A05258-3). Assess cytoplasmic staining by cell type, allowing for a granular pattern in some tissues (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
AFAP1 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 (HPA tissue IHC); stress fibers expected (UniProt) | |
| Staining pattern | Cytoplasmic, sometimes granular; high in appendix endocrine cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A05258-3) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
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 A05258-3) | |
| Caveat | Antibody staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Overexpressed in prostate carcinoma (UniProt) | |
| Isoform / epitope | 2 isoforms; verify antibody epitope coverage (UniProt) |
Section 1
Recommended AFAP1 IHC & IF Protocols
The catalog antibody uses heat-mediated EDTA retrieval at pH 8.0 (datasheet A05258-3). One published AFAP1 IHC protocol provides a comparison for human paraffin sections (PMC3085893).
| Sample | Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet A05258-3) |
| Fixation | Image fixative and duration unreported (datasheet A05258-3); 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 A05258-3); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A05258-3) |
| Primary antibody | Rabbit anti-AFAP1, 2-5μg/ml (datasheet A05258-3) |
| Primary incubation | Overnight at 4 °C (datasheet A05258-3) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A05258-3) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | AFAP1-positive staining in endocrine cells of appendix (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression which a granular pattern in several tissues. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 for the catalog antibody (datasheet A05258-3); the published F1 antibody protocol used citrate pH 6.0 (PMC3085893).
Section 2
What Is the Expected AFAP1 Staining Pattern?
AFAP1 is a cytoplasmic, actin-associated protein with no transmembrane segment (UniProt Q8N556). In paraffin-section IHC, expect cytoplasmic staining that may appear granular across several tissues (HPA: tissue IHC, Enhanced reliability). Appendix endocrine cells and gallbladder glandular cells are useful strong examples; staining can also occur in other cell types (HPA: High in both; ubiquitous cytoplasmic profile).
| Granular cytoplasmic staining in appendix endocrine cells or gallbladder glandular cells, with recognizable cell boundaries. | This fits the reported compartment and High staining in those cells (HPA: tissue IHC). Interpret intensity by cell type; the reported profile is widespread, so other cytoplasmic staining is possible (HPA: ubiquitous cytoplasmic expression). |
| Strong nuclear-only staining, or signal confined to extracellular material, without convincing cytoplasmic staining. | Treat this as discordant with the expected AFAP1 location, rather than evidence of a new compartment (UniProt Q8N556: cytoplasm, cytoskeleton, stress fiber; HPA: cytoplasmic IHC profile). Review the control and detection conditions (general IHC practice). |
| The strongest signal is in a cell population reported as Low, while expected High cells show little staining. | A swapped cell-type pattern raises concern for cross-reactivity or endogenous detection activity (general IHC interpretation). For example, compare thyroid glandular cells, reported Low, with gallbladder glandular cells, reported High (HPA: tissue IHC); Low does not mean absent. |
| A uniform brown haze covers cells, stroma, or section edges and obscures cell boundaries. | Diffuse background cannot establish AFAP1 localisation (general IHC interpretation). Compare a reagent control and inspect whether signal follows cytoplasm in identifiable cells; the expected tissue profile is cytoplasmic, sometimes granular (HPA: tissue IHC). |
| No convincing signal appears in appendix endocrine cells or gallbladder glandular cells. | These are reported High examples, so absence there makes a negative study result hard to interpret (HPA: tissue IHC). Check section identity and run performance before assigning a biological absence (general IHC practice). |
💡Expected AFAP1 appearanceCall a result positive when identifiable appendix endocrine or gallbladder glandular cells show clear, often granular High cytoplasmic staining (HPA: tissue IHC); isolated nuclear colour or diffuse, cell-independent colour is suspect (UniProt Q8N556: cytoplasmic localisation; general IHC interpretation).
| Subcellular location | AFAP1 is associated with the cytoskeleton and stress fibers and has no transmembrane segment (UniProt Q8N556). Judge IHC by cytoplasmic localisation; a membrane outline alone does not match the supplied location evidence. |
| Tissue and cell-type context | HPA reports ubiquitous cytoplasmic expression with a granular pattern in several tissues, but levels differ by cell type (HPA: tissue IHC). Use the named High cells as reference points; do not expect every cell to match their intensity. |
| Antibody validation | Two listed antibodies have Enhanced IHC status, HPA015642 and CAB024712 (HPA: antibody validation). The overall tissue profile has Enhanced reliability with medium agreement between staining and RNA, so resolve discordant fields with controls (HPA: tissue IHC). |
| IF/ICC comparison | IF/ICC images place AFAP1 mainly at actin filaments, with focal adhesion sites additionally approved and cytosol uncertain (HPA: subcellular ICC-IF). This supports a cytoplasmic interpretation, but the IF/ICC evidence is not a paraffin-section protocol. |
| Isoforms and modification | UniProt lists 2 isoforms and multiple modified residues (UniProt Q8N556). The supplied sources do not establish how those features change this antibody's IHC staining; do not infer an isoform-specific or phosphorylation-specific pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| The expected High cells are unstained. | The run may have failed, the reference cells may be absent from the section, or the detection conditions may be unsuitable (general IHC practice). | Confirm the cell types on the counterstain, then assess a known-positive section and the run controls before interpreting absence (general IHC practice; HPA: High in appendix endocrine and gallbladder glandular cells). |
| All compartments show a diffuse brown haze. | Nonspecific reagent binding or detection background may obscure the expected cellular pattern (general IHC practice; HPA: cytoplasmic IHC profile). | Review blocking, washing, reagent controls, and chromogen development within the established IHC workflow (general IHC practice). Reassess only signal that resolves to identifiable cytoplasm (HPA: tissue IHC). |
| Nuclei are strongly stained while cytoplasm is weak. | A dominant nuclear-only pattern conflicts with AFAP1's recorded localisation (UniProt Q8N556: cytoplasm, cytoskeleton, stress fiber). | Check the counterstain and detection controls, then compare with a known-positive section for cytoplasmic signal (general IHC practice; HPA: cytoplasmic tissue profile). |
| Low-reference cells dominate the slide. | Cross-reactivity or endogenous detection activity is possible when the observed cell ranking disagrees with the reference pattern (general IHC interpretation; HPA: tissue IHC levels). | Compare a High-reference cell type in the same run and inspect reagent controls (general IHC practice). Thyroid glandular cells are reported Low, while gallbladder glandular cells are High (HPA: tissue IHC). |
| The signal appears only as crisp cell-membrane outlines. | A membrane-only readout is discordant with the supplied cytoplasmic and actin-associated location evidence (UniProt Q8N556; HPA: tissue IHC and subcellular ICC-IF). | Recheck compartment assignment at higher magnification and compare a control section with identifiable cytoplasm (general IHC practice). Avoid scoring membrane outlines alone as AFAP1-positive. |
| IHC looks different from an IF/ICC image. | The HPA IF/ICC summary describes actin filaments and focal adhesions in imaged cells, whereas tissue IHC reports a broader cytoplasmic, sometimes granular pattern (HPA: subcellular ICC-IF; tissue IHC). | Score the paraffin section against its tissue and cell-type IHC reference (HPA: tissue IHC). Use the IF/ICC location as supporting context, without treating it as an IHC protocol or requiring identical visual detail. |
Experiment evidence
IHC & IF images showing AFAP1 staining
A05258-3 · IHCIHC analysis of AFAP/AFAP1 using anti-AFAP/AFAP1 antibody (A05258-3). AFAP/AFAP1 was detected in paraffin-embedded section of human lung can
A05258-3 · IHCIHC analysis of AFAP/AFAP1 using anti-AFAP/AFAP1 antibody (A05258-3). AFAP/AFAP1 was detected in paraffin-embedded section of human prostati
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000196526-AFAP1/tissue/cerebral+cortex#imid_7609960
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000196526-AFAP1/tissue/colon#imid_7609886
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000196526-AFAP1/tissue/liver#imid_7609879
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000196526-AFAP1/tissue/kidney#imid_7609888
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA015642; green = AFAP1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA015642; green = AFAP1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for AFAP1 IHC & IF
🧪Run appendix first and look for staining in endocrine cells (HPA: appendix endocrine cells High). HPA detects AFAP1 in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype slides to define background, and treat unstained cells within the appendix section as internal background comparators rather than confirmed AFAP1-negative cells (HPA: no negative tissue rows).
Positive control tissue: Appendix (Endocrine cells, HPA High)
Negative control tissue: None in HPA: AFAP1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show AFAP1 in A-431, U-251MG, U2OS, with annotated localisation: Actin filaments (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only section, a concentration-matched rabbit IgG isotype control, and AFAP1-knockout material as a biological negative where available (selected-SKU caption: rabbit primary antibody). Because the reported chromogenic method uses a biotinylated secondary, SABC and DAB, check endogenous peroxidase and biotin background in the positive section (selected-SKU caption: SABC/DAB detection).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU caption: fixative unreported). That caption reports heat retrieval in EDTA at pH 8.0 for lung cancer tissue, but does not establish its necessity for appendix (selected-SKU caption: EDTA retrieval in lung cancer tissue). ICC-IF images support actin-filament localization in A-431, U-251MG and U2OS cells, but the supplied evidence does not establish that IF or frozen sections are easier than paraffin IHC or identify an appendix-specific artefact (HPA subcellular: actin filaments supported; selected-SKU caption: paraffin section).
HPA tissue IHC evidence for AFAP1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — 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 |
|---|---|---|---|---|
| Appendix | Endocrine cells | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Adipose tissue | Adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: AFAP1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced AFAP1 IHC Tips
Use the catalog antibody’s paraffin-section workflow as the starting point, then assess AFAP1 staining by cell type, compartment and controls.
Which antigen retrieval should I use for AFAP1 in paraffin sections?
Start with heat-mediated EDTA retrieval at pH 8.0 for paraffin sections (datasheet A05258-3). The selected tissue image used this retrieval before incubation with 2 µg/ml primary antibody overnight at 4°C, so keep those conditions together when establishing a baseline (caption A05258-3). If staining is weak, compare a second retrieval condition on adjacent sections while holding antibody concentration and detection constant; retrieval changes can alter both signal and tissue morphology (standard IHC practice). Record heating and cooling conditions for each run, since the caption specifies neither duration nor equipment (caption A05258-3). Judge improvement by cytoplasmic staining in expected cells and by background on matched controls (UniProt Q8N556 subcellular location; standard IHC practice).
How should I handle fixation when AFAP1 staining is inconsistent?
AFAP1-specific fixation sensitivity is unknown from the supplied evidence: the selected paraffin-section caption does not state a fixative (caption A05258-3). Record fixative, fixation duration, processing history and section age for each specimen before comparing staining intensity (standard IHC practice). Establish a reference run using EDTA retrieval at pH 8.0 and 2 µg/ml primary antibody overnight at 4°C, as reported for this antibody (caption A05258-3). If specimens differ in fixation history, compare matched sections and assess morphology alongside staining rather than assigning every intensity difference to AFAP1 abundance (standard IHC practice). HPA staining patterns and AFAP1 topology do not establish a fixation response (HPA tissue IHC; UniProt Q8N556 topology).
Where should AFAP1 signal appear in chromogenic tissue sections?
Prioritise cytoplasmic staining when reading AFAP1 IHC: HPA describes ubiquitous cytoplasmic expression, often with a granular pattern (HPA tissue IHC). AFAP1 is associated with cytoskeletal stress fibers, and subcellular imaging supports actin-filament localisation with additional focal-adhesion localisation (UniProt Q8N556 subcellular location; HPA subcellular). Fine actin structures may be difficult to resolve with chromogenic tissue staining, so score the visible cellular compartment rather than requiring filament-shaped DAB deposits (standard IHC practice). Compare epithelial, stromal and other cells separately within the same section, because tissue-level staining can reflect cell composition (standard IHC practice). Treat isolated nuclear or extracellular deposits as suspect until morphology and controls support them (UniProt Q8N556 subcellular location; standard IHC practice).
Can this stain distinguish AFAP1 isoforms or phosphorylation states?
Do not assign staining to a particular isoform without epitope mapping: AFAP1 has 2 recorded isoforms, while the supplied antibody caption does not specify an epitope (UniProt Q8N556 isoforms; caption A05258-3). AFAP1 also has annotated phosphoserines, including residues 282, 283, 548 and 664, but no supplied evidence establishes phosphorylation-selective recognition (UniProt Q8N556 modified residues; caption A05258-3). Compare sections processed together when assessing differences, because retrieval and detection affect apparent intensity (standard IHC practice). If an isoform-specific conclusion matters, obtain the antibody’s epitope information and validate discrimination with an appropriate reference material (standard IHC practice). Report the result as AFAP1 immunoreactivity until that specificity is established (standard IHC practice).
How can I investigate AFAP1 localisation with multiplex IF?
Use this as a separate IF optimisation: the selected antibody evidence describes chromogenic staining of paraffin sections, not IF performance (caption A05258-3). Pair AFAP1 with an independently validated marker for the cell population being assessed, such as the glandular cells under comparison, and verify each channel alone before multiplexing (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores and place the weaker signal in a channel with less tissue autofluorescence; include an unstained section to assess that background (standard IF practice). Because AFAP1 is cytoplasmic and has no transmembrane segment, use permeabilisation appropriate for an intracellular epitope and compare its effect on morphology (UniProt Q8N556 topology and subcellular location; standard IF practice). HPA reports actin-filament localisation that IF can help assess (HPA subcellular).
What should I check when AFAP1 DAB staining is diffuse?
First compare the stained section with a no-primary control and inspect whether deposits follow cellular morphology or appear across tissue and blank areas (standard IHC practice). The selected image used 10% goat serum blocking, a biotinylated secondary antibody and a streptavidin–biotin detection system with DAB (caption A05258-3). Check endogenous peroxidase blocking and, for this detection system, assess endogenous biotin contribution with a suitable control (standard IHC practice; caption A05258-3). If background persists, review washing, blocking and antibody concentration while retaining the documented 2 µg/ml condition as the comparison point (caption A05258-3; standard IHC practice). AFAP1 can show granular cytoplasmic staining, so evaluate whether granules remain cell-associated before calling them background (HPA tissue IHC).
How should I quantify AFAP1 staining across tissue samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue compartment and cell population before scoring, then apply the same inclusion rules to every section (standard IHC practice). For cellular staining, report the percentage of positive cells and an H-score combining intensity with that percentage; state the intensity thresholds and score range used (standard IHC practice). If counting discrete positive cells, report density per mm² of viable tissue and exclude folds, necrosis and blank regions consistently (standard IHC practice). Normalise comparisons to the same eligible cell population or viable tissue area, and keep retrieval, detection and imaging conditions comparable across samples (standard IHC practice). HPA reports low tissue specificity and medium staining–RNA consistency, so document cell composition when interpreting differences (HPA tissue IHC).
How do I distinguish convincing AFAP1 staining from artefact?
A convincing result is cell-associated cytoplasmic staining with interpretable morphology, consistent with AFAP1’s cytoskeletal location and HPA’s granular cytoplasmic tissue pattern (UniProt Q8N556 subcellular location; HPA tissue IHC). Evaluate the identity of stained cells: HPA reports high staining in appendix endocrine cells and gallbladder glandular cells, but low staining in bronchial respiratory epithelial cells (HPA tissue IHC). Reject edge-only signal, staining concentrated in necrosis and deposits without cell boundaries as likely artefacts pending controls (standard IHC practice). Use a no-primary control and check endogenous enzyme activity when DAB is widespread, since the selected workflow uses chromogenic detection (standard IHC practice; caption A05258-3). Interpret differences cautiously because HPA reports only medium agreement between antibody staining and RNA expression (HPA tissue IHC).
Boster reagents
Best AFAP1 / Actin filament-associated protein 1 IHC Antibodies
A05258-3 has IHC images from paraffin-embedded human lung and prostatic cancer sections (A05258-3 image captions). Human, mouse and rat reactivity is listed (A05258-3 catalog).
Real IHC data
Anti-AFAP/AFAP1 Antibody ®
Cat # A05258-3
A05258-3 was demonstrated by IHC on paraffin-embedded human lung and prostatic cancer sections (A05258-3 image captions). Its catalog lists IHC, but provides no IF/ICC application or IF image (A05258-3 catalog).
Which to pick: Choose A05258-3 for human paraffin-section IHC because its own captions document staining in those sections; the fixative is unreported (A05258-3 image captions). No SKU here has documented IF/ICC use, so IF/ICC staining requires separate validation (A05258-3 catalog). For mouse or rat work, A05258-3 lists both species as reactive, but its documented IHC examples and 2–5 μg/ml IHC recommendation are for human samples; verify staining in the intended species (A05258-3 catalog; A05258-3 image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8N556 (AFAP1_HUMAN, Actin filament-associated protein 1).
- Human Protein Atlas. AFAP1 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. AFAP1 subcellular location (ICC-IF): Mainly localized to the actin filaments. In addition localized to the cytosol and focal adhesion sites..
- Human Protein Atlas. AFAP1 antibody validation summary (2 antibodies).
- Co-expression of AFAP1-AS1 and PD-1 predicts poor prognosis in nasopharyngeal carcinoma. Oncotarget 2017 — PMC5503590.
- AFAP1L1 is a novel adaptor protein of the AFAP family that interacts with cortactin and localizes to invadosomes. European journal of cell biology 2011 — PMC3085893.
- Long noncoding RNA actin filament-associated protein 1 antisense RNA 1 promotes malignant phenotype through binding with lysine-specific demethylase 1 and repressing HMG box-containing protein 1 in non-small-cell lung cancer. Cancer science 2019 — PMC6609801.
- Long non-coding RNA AFAP1-AS1 promotes alternative splicing of AXIN2 by facilitating SRSFs phase separation to induce drug resistance in lung adenocarcinoma. Molecular cancer 2025 — PMC12632050.
- PubMed PMID:15485829 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15815621 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for AFAP1 (Actin filament-associated protein 1). Expected localisation Cytoplasm observed (HPA tissue IHC); stress fibers expected (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A05258-3). Validated IHC antibody: A05258-3 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Endocrine cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet A05258-3); 2. Fixation: Image fixative and duration unreported (datasheet A05258-3); 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 A05258-3); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A05258-3); 8. Primary antibody: Rabbit anti-AFAP1, 2-5μg/ml (datasheet A05258-3); 9. Primary incubation: Overnight at 4 °C (datasheet A05258-3). 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 A05258-3); Caveat: Antibody staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 2 isoforms; verify antibody epitope coverage (UniProt); Regulation: Overexpressed in prostate carcinoma (UniProt). Evidence: UniProt Q8N556, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the actin filaments. In addition localized to…, Datasheet A05258-3, PMC3085893. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/AFAP1/immunohistochemistry-ihc-guide.