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WASF2 / Actin-binding protein WASF2 · IHC design guide
Design Immunohistochemistry for WASF2
Plan paraffin section IHC for WASF2 using its reported cytoplasmic tissue staining and high staining in selected glandular and neuronal cells (HPA tissue IHC). The guide covers fixation consistency, staining interpretation and controls.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
WASF2 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 tissue staining (HPA tissue IHC); lamellipodia are a molecular location (UniProt) | |
| Staining pattern | High cytoplasmic staining in glandular and neuronal cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| 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 | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M03969) | |
| Caveat | Staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | No expression regulator specified (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage is undetermined (UniProt) |
Section 1
Recommended WASF2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by a published gastric cancer tissue protocol (PMC5497796).
| Sample | Paraffin-embedded human colon tissue; fixative not specified (datasheet M03969) |
| Fixation | Image fixative and duration unreported (datasheet M03969); 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 monoclonal (clone FFG-23) anti-WASF2, 1:50 (datasheet M03969) |
| 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 | WASF2-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval rule); the published protocol also uses microwave-heated citrate (PMC5497796).
Section 2
What Is the Expected WASF2 Staining Pattern?
WASF2 should show mainly cytoplasmic staining in paraffin sections (HPA: Enhanced reliability; general cytoplasmic expression). High staining is reported in glandular cells of appendix, breast, colon, duodenum and gallbladder, and neuronal cells of caudate, cerebral cortex and hippocampus (HPA: tissue IHC). WASF2 also associates with the cytoskeleton, lamellipodia and basolateral membrane (UniProt Q9Y6W5: subcellular location); it has no transmembrane segment (UniProt Q9Y6W5: topology).
| Cytoplasmic signal in glandular or neuronal cells, with occasional edge accentuation. | This fits the reported general cytoplasmic IHC pattern and high staining in the listed glandular and neuronal populations (HPA: tissue IHC). Edge accentuation is plausible from WASF2's lamellipodial and basolateral membrane locations (UniProt Q9Y6W5: subcellular location), but HPA tissue IHC does not establish a required membrane pattern. |
| Predominantly nuclear, luminal or extracellular deposit with little cytoplasmic staining. | Treat an isolated wrong-compartment pattern as suspect: the expected IHC pattern is cytoplasmic (HPA: tissue IHC), and UniProt places WASF2 in the cytoskeleton and cell projections (UniProt Q9Y6W5: subcellular location). Review morphology, counterstain and detection controls before scoring it as specific (general IHC practice). |
| Strong staining in cells expected to be low, while the expected positive cells are weak. | Ovarian stroma cells, soft-tissue fibroblasts and splenic white-pulp cells are reported low, not definitively negative (HPA: tissue IHC). Unexpected strong signal can reflect cross-reactivity or endogenous detection activity (general IHC practice). Compare cell identity and control sections; a low HPA level alone cannot prove that staining is false. |
| Diffuse color across tissue, stroma and blank areas, obscuring cell boundaries. | A broad deposit is difficult to reconcile with a cell-associated cytoplasmic pattern (HPA: tissue IHC). Nonspecific antibody binding, inadequate washing or endogenous detection activity can raise background (general IHC practice). Judge specificity only after background is reduced and the expected cells remain distinguishable. |
| No signal in a section containing a reported high-staining cell population. | Absence in appendix or colon glandular cells, for example, conflicts with the reported high IHC pattern (HPA: tissue IHC). First assess tissue preservation and the staining run with appropriate controls (general IHC practice). A single negative section does not establish that WASF2 is absent from that tissue. |
💡Expected WASF2 appearanceCall positive a clear, mainly cytoplasmic signal in the reported high-staining glandular or neuronal cells (HPA: tissue IHC), allowing possible cell-edge accentuation (UniProt Q9Y6W5: subcellular location); isolated nuclear or diffuse acellular color is suspect (general IHC practice).
| Tissue and cell population | HPA reports high staining in specified glandular and neuronal cells, but low staining in ovarian stroma, soft-tissue fibroblasts and splenic white pulp (HPA: tissue IHC). Compare the same cell population across sections; these ratings are not universal cutoffs. |
| Antibody validation | HPA045288 has Enhanced IHC status and CAB022632 has Supported IHC status (HPA: antibody validation). HPA describes its overall tissue result as Enhanced with medium consistency between antibody staining and RNA expression (HPA: tissue IHC); interpret borderline staining cautiously. |
| Isoforms and modification | UniProt lists two isoforms and phosphoserine at residue 474 (UniProt Q9Y6W5: isoforms; modified residues). The payload gives no antibody epitope or evidence that these features alter IHC staining, so neither predicts a section-level intensity difference. |
| Can IF/ICC show a different distribution? | Yes. HPA reports approved plasma-membrane localization with additional cytosol localization in ICC-IF (HPA: subcellular; HPA045288 ICC Approved). That observation supports a localization comparison, but it does not turn an IF image into an IHC-P protocol or require crisp membrane staining in chromogenic sections. |
| Situation | Likely cause | Next action |
|---|---|---|
| No staining in expected positive cells. | The run may have failed, or the sampled section may lack the relevant cells (general IHC practice). | Check morphology and a contemporaneous positive control; use a section with a reported high-staining population, such as colon glandular cells (HPA: tissue IHC). Review the catalog antibody's IHC-P instructions and the run's retrieval and detection records (general IHC practice). |
| Nuclear staining dominates. | Nuclear localization is outside the reported general cytoplasmic IHC pattern (HPA: tissue IHC). | Verify that counterstain is not being mistaken for chromogen, then compare with a detection control and a reported positive tissue (general IHC practice; HPA: tissue IHC). Do not score an isolated nuclear pattern as expected WASF2. |
| Strong staining appears in reported low-staining cells. | Cross-reactivity or endogenous detection activity is possible (general IHC practice); low HPA staining does not mean zero protein (HPA: tissue IHC). | Confirm cell identity and compare a no-primary control (general IHC practice). If the signal tracks the detection control, address the detection background; if it persists only with primary antibody, assess antibody specificity. |
| Color is widespread and cell borders are unclear. | Nonspecific binding, excess reagent or insufficient washing can obscure a cellular pattern (general IHC practice). | Inspect blank areas and a no-primary control, then optimize blocking, antibody concentration and washes within the validated IHC-P workflow (general IHC practice). Reassess whether cytoplasmic staining remains in the expected cells (HPA: tissue IHC). |
| Only membrane staining is visible. | WASF2 can associate with the basolateral membrane (UniProt Q9Y6W5: subcellular location), while HPA describes general cytoplasmic tissue staining (HPA: tissue IHC). | Check whether cytoplasm is also labeled and whether signal follows intact cells (general IHC practice). Compare with a reported high-staining tissue (HPA: tissue IHC); an isolated membrane rim is insufficient to establish the expected IHC pattern. |
| Staining varies sharply between adjacent sections. | Differences in sampled cell populations or staining runs can change the apparent result (general IHC practice). HPA reports cell-specific levels and medium antibody/RNA consistency (HPA: tissue IHC). | Compare matched regions and controls before assigning a biological difference (general IHC practice). Record the cell population and compartment scored, and treat borderline changes cautiously (HPA: tissue IHC). |
Experiment evidence
IHC & IF images showing WASF2 staining
M03969 · IHCImmunohistochemical analysis of paraffin-embedded human colon, using WASF2 Antibody.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000158195-WASF2/tissue/cerebral+cortex#imid_6678704
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000158195-WASF2/tissue/colon#imid_6678638
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000158195-WASF2/tissue/liver#imid_6678623
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000158195-WASF2/tissue/kidney#imid_6678624
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA045288; green = WASF2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA045288; green = WASF2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for WASF2 IHC & IF
🧪Run human colon first; its glandular cells should stain (HPA: High in colon glandular cells; selected SKU M03969: paraffin-section colon image). HPA detects WASF2 in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and expect little staining in tissue spaces or other structures without cells on the positive slide (HPA: no negative tissue listed; standard IHC background assessment).
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: None in HPA: WASF2 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 WASF2 in A-431, HEK293, U2OS, NIH 3T3, with annotated localisation: Plasma membrane (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide and an isotype control matched to the primary antibody’s host species and clonality; use WASF2 knockout material or a peptide-block control if the immunizing peptide is available (standard IHC specificity controls). Check endogenous peroxidase signal in colon sections before interpreting chromogenic staining (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and its effect on WASF2 staining are unreported, and the selected SKU M03969 colon caption does not state a fixative (selected SKU M03969 caption). Retrieval dependence is unreported; optimize antigen retrieval on paraffin sections empirically (standard IHC practice). The supplied evidence does not establish that frozen sections or IF are easier; assess colon background from endogenous peroxidase in IHC and autofluorescence if using IF (standard IHC/IF practice).
HPA tissue IHC evidence for WASF2
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 | Glandular cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: WASF2 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced WASF2 IHC Tips
Troubleshoot WASF2 staining by checking retrieval, compartment, cell type and controls before comparing chromogenic IHC results across sections.
How should I retrieve WASF2 in paraffin sections when the signal is weak?
Use citrate buffer at pH 6.0 for heat-induced retrieval at 95–98 °C for 20 minutes (page retrieval rule). Cool sections in the retrieval buffer, then rinse and compare a known positive glandular area with a no-primary control (HPA: High in colon glandular cells; standard IHC practice). The catalog antibody has a paraffin-section image from human colon, although its caption does not specify the fixative (SKU M03969 tissue-IHC caption). If signal remains weak, compare a modestly longer retrieval time on adjacent sections while monitoring tissue damage (standard IHC practice). Keep detection and exposure conditions matched so a retrieval change is interpretable (standard IHC practice).
Could fixation explain weak or uneven WASF2 staining in my paraffin sections?
Target-specific WASF2 sensitivity to fixation is unknown from the supplied evidence, and the catalog image identifies paraffin-embedded human colon without naming its fixative (SKU M03969 tissue-IHC caption). Record the actual fixative, fixation duration and processing history for each specimen before comparing intensity (standard IHC practice). On adjacent sections, hold citrate retrieval at pH 6.0, 95–98 °C and 20 minutes constant while varying only the documented processing condition (page retrieval rule; standard IHC practice). Inspect morphology and include the same positive control with every run (standard IHC practice). Neither the tissue expression pattern nor WASF2 topology establishes a fixation effect (HPA: general cytoplasmic expression; UniProt Q9Y6W5: no transmembrane segment).
Should WASF2 appear at cell borders or throughout the cytoplasm?
Expect predominantly cytoplasmic staining in tissue IHC, with possible enrichment near cell edges, because the tissue profile reports general cytoplasmic expression (HPA: tissue IHC profile). WASF2 is associated with the cytoskeleton, lamellipodia and the interface between lamellipodial actin and membrane (UniProt Q9Y6W5: subcellular location). Approved cell-imaging annotations also place it mainly at the plasma membrane and additionally in cytosol (HPA: subcellular). Assess border accentuation against intact cell morphology and adjacent cytoplasm at the same magnification (standard IHC practice). Diffuse nuclear-only staining needs independent validation before assignment to WASF2 (UniProt Q9Y6W5: listed locations; standard IHC practice).
How do isoforms and epitope position affect interpretation of WASF2 staining?
WASF2 has 2 listed isoforms, but the supplied catalog evidence does not locate this antibody's epitope or establish isoform coverage (UniProt Q9Y6W5: isoforms; SKU M03969 tissue-IHC caption). Its WH2 domain spans residues 436–453, and a reported phosphoserine occurs at residue 474 (UniProt Q9Y6W5: domains and modified residues). Check the antibody's disclosed immunogen sequence against both isoforms before interpreting a negative region as absent protein (standard IHC practice). Because WASF2 has no transmembrane segment, describe any border staining as peripheral localisation rather than proof of a membrane-spanning epitope (UniProt Q9Y6W5: topology and subcellular location). Keep retrieval and detection identical across sections used for that comparison (standard IHC practice).
How can I check a chromogenic WASF2 pattern with multiplex immunofluorescence?
Use IF as an orthogonal localisation check, pairing WASF2 with a glandular-cell marker selected for the specimen, since colon glandular cells show high tissue staining (HPA: High in colon glandular cells). Choose fluorophores after measuring unstained-section autofluorescence, and reserve the cleaner spectral channel for the weaker signal (standard IF practice). For an intracellular epitope, compare gentle permeabilisation with an unpermeabilised section; WASF2 lacks a transmembrane segment and is associated with cytosol and peripheral actin (UniProt Q9Y6W5: topology and subcellular location). Include single-stain and no-primary controls to assess bleed-through and background (standard IF practice). IF processing and antibody suitability require separate validation from the paraffin IHC caption (SKU M03969 tissue-IHC caption; standard IF practice).
What should I check when WASF2 DAB staining is widespread?
First compare a no-primary section with the stained section to identify detection-system background (standard IHC practice). For chromogenic peroxidase detection, apply an appropriate peroxide block before DAB development and compare the resulting background on matched sections (standard IHC practice). Reduce excessive primary-antibody concentration or incubation if background rises across cells and extracellular areas together; optimise one variable at a time (standard IHC practice). WASF2 has broad tissue expression and a general cytoplasmic IHC profile, so widespread cellular signal alone does not establish nonspecific staining (UniProt Q9Y6W5: tissue specificity; HPA: tissue IHC profile). Examine folds, section edges and damaged regions separately from intact tissue (standard IHC practice).
How should I score WASF2 staining across colon sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the analysis compartment as morphologically intact glandular cells, which have high reported staining in colon (HPA: High in colon glandular cells; standard IHC practice). Record the percentage of positive cells and intensity categories, then calculate an H-score as the sum of each intensity score multiplied by its cell percentage (standard IHC practice). Normalise comparisons to the number of evaluable glandular cells or glandular area, and report sampled fields and excluded damaged areas (standard IHC practice). Use matched retrieval at pH 6.0, 95–98 °C for 20 minutes and consistent DAB development across groups (page retrieval rule; standard IHC practice). Keep thresholds fixed using controls from the same run (standard IHC practice).
How do I separate genuine WASF2 positivity from staining artefact?
A credible pattern follows intact cell contours or cytoplasm rather than appearing only in nuclei or extracellular debris (UniProt Q9Y6W5: subcellular location; HPA: tissue IHC profile; standard IHC practice). In colon, compare glandular cells with neighbouring structures because glandular cells have high reported staining (HPA: High in colon glandular cells). Treat edge-only signal, necrotic areas and pooled DAB as suspect until the same pattern appears in well-preserved tissue (standard IHC practice). Check no-primary and peroxide-block controls for endogenous-enzyme or detection background before assigning weak positivity (standard IHC practice). Interpret differences cautiously because the tissue atlas reports medium consistency between antibody staining and RNA expression (HPA: Enhanced reliability description).
Boster reagents
Best WASF2 / Actin-binding protein WASF2 IHC Antibodies
The IHC-validated anti-WASF2 antibody has a paraffin-section image from human colon; catalog reactivity includes human, mouse, and rat (catalog applications; image caption; reactivity). No IF data are supplied (catalog image list).
Real IHC data
Anti-WASF2 Rabbit Monoclonal Antibody
Cat # M03969
M03969 is listed for IHC and has an image caption describing paraffin-embedded human colon (catalog applications; image caption). Its listed reactivity is human, mouse, and rat; no IF image or IF application is supplied (catalog reactivity; applications; image list).
Which to pick: Choose M03969 for paraffin-section tissue IHC: it is a rabbit monoclonal with an IHC image from human colon (catalog title; image caption). Its catalog reactivity includes mouse and rat, but the supplied IHC image documents human tissue only (catalog reactivity; image caption). No listed SKU is validated for IF/ICC, and the fixative used for the paraffin section is unreported (catalog applications; image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9Y6W5 (WASF2_HUMAN, Actin-binding protein WASF2).
- Human Protein Atlas. WASF2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. WASF2 subcellular location (ICC-IF): Mainly localized to the plasma membrane. In addition localized to the cytosol..
- Human Protein Atlas. WASF2 antibody validation summary (2 antibodies).
- Clinicopathological significance of the microRNA-146a/WASP-family verprolin-homologous protein-2 axis in gastric cancer. Cancer science 2017 — PMC5497796.
- WASF2 Serves as a Potential Biomarker and Therapeutic Target in Ovarian Cancer: A Pan-Cancer Analysis. Frontiers in oncology 2022 — PMC8964075.
- Hypomethylation-mediated upregulation of the WASF2 promoter region correlates with poor clinical outcomes in hepatocellular carcinoma. Journal of experimental & clinical cancer research : CR 2022 — PMC9047373.
- Efficient delivery of small interfering RNAs targeting particular mRNAs into pancreatic cancer cells inhibits invasiveness and metastasis of pancreatic tumors. Oncotarget 2019 — PMC6499602.
- PubMed PMID:10381382 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16710414 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for WASF2 (Actin-binding protein WASF2). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC); lamellipodia are a molecular location (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: M03969 at 1:50 (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon tissue; fixative not specified (datasheet M03969); 2. Fixation: Image fixative and duration unreported (datasheet M03969); 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 monoclonal (clone FFG-23) anti-WASF2, 1:50 (datasheet M03969); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M03969); Caveat: Staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage is undetermined (UniProt); Regulation: No expression regulator specified (UniProt). Evidence: UniProt Q9Y6W5, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the plasma membrane. In addition localized to…, Datasheet M03969, PMC5497796. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/WASF2/immunohistochemistry-ihc-guide.