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WTAP / Pre-mRNA-splicing regulator WTAP · IHC design guide
Design Immunohistochemistry for WTAP
Plan chromogenic WTAP IHC in paraffin sections with the catalog antibody at 1:50–1:200 (datasheet). Use colon glandular cells with high staining as a positive reference and assess nuclear intensity across cell types (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
WTAP 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 | Nuclear staining in tissue sections (HPA tissue IHC) | |
| Staining pattern | Widespread nuclear staining at variable intensity (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Heart muscle |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Cardiomyocytes may show no detectable staining (HPA tissue IHC) | |
| Regulation | Ubiquitous expression; variable tissue levels (UniProt; HPA tissue IHC) | |
| Isoform / epitope | 2 isoforms; epitope coverage is unclear (UniProt) |
Section 1
Recommended WTAP IHC & IF Protocols
Compare the catalog antibody's IHC-P protocol (datasheet) with published WTAP IHC protocols for ovarian tissue (PMC6689666) and glioblastoma tissue (PMC11006471).
| Sample | Paraffin-embedded human tonsil cancer tissue; fixative not specified (datasheet A04296-1) |
| Fixation | Image fixative and duration unreported (datasheet A04296-1); 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 Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear 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-WTAP, 1:50-1:200 (datasheet A04296-1) |
| 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 | WTAP-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear expression at variable levels. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 HIER at 95–98 °C for 20 min (page retrieval rule); citrate retrieval at 100 °C for 10 min is a published alternative (PMC6689666).
Section 2
What Is the Expected WTAP Staining Pattern?
WTAP should stain mainly nuclei, with possible weaker cytoplasmic signal (UniProt Q15007: subcellular location). Nuclear staining varies across tissues and cell types; HPA reports high staining in several glandular, hematopoietic, respiratory epithelial, and neuronal populations (HPA: tissue IHC). Treat this as a supported pattern with medium consistency between antibody staining and RNA expression (HPA: reliability). WTAP has no transmembrane segment (UniProt Q15007: topology).
| Nuclear staining predominates in glandular cells of colon, breast, or adrenal gland; intensity varies across cells. | This fits the expected compartment and examples of high staining (UniProt Q15007: mainly nuclear; HPA: High in these glandular cells). Score the nuclei of the named cell population, since HPA describes variable levels across tissues (HPA: ubiquitous nuclear expression). |
| Staining appears concentrated in subnuclear puncta, with little cytoplasmic signal. | Nuclear speckles are consistent with WTAP localisation (UniProt Q15007: nuclear speckle; HPA: enhanced nuclear-speckle location by ICC-IF). Speckles may be easier to distinguish by fluorescence microscopy; a chromogenic IHC result can still be consistent when its dominant signal is nuclear (general IHC interpretation). |
| Strong membrane staining or cytoplasmic staining dominates while nuclei are faint. | This conflicts with the mainly nuclear distribution and lack of a transmembrane segment (UniProt Q15007: subcellular location and topology). Some cytoplasmic WTAP is possible (UniProt Q15007: subcellular location), so assess whether nuclear staining remains predominant before treating the slide as an artefact. |
| Cardiomyocytes stain strongly, or chromogen appears in cells inconsistent with the expected tissue pattern. | HPA reports WTAP as not detected in heart-muscle cardiomyocytes (HPA: tissue IHC). Unexpected cell staining warrants checks for antibody cross-reactivity or endogenous detection activity (general IHC practice); this single HPA observation is a comparison point, not an absolute biological absence claim (HPA: Supported, medium consistency). |
| The entire section has diffuse colour, or a known high-staining population has no nuclear signal. | Diffuse colour limits compartment scoring and may reflect background from antibody, detection reagents, or endogenous activity (general IHC practice). No nuclear signal in a high-staining reference population, such as bone-marrow hematopoietic cells, makes the run inconclusive (HPA: High in hematopoietic cells; general IHC practice). |
💡Expected WTAP appearanceA convincing positive is predominantly nuclear chromogen in the expected cells, potentially strong in colon glandular cells or bone-marrow hematopoietic cells (UniProt Q15007: mainly nuclear; HPA: High in both); diffuse colour or dominant membrane staining does not match that localisation (UniProt Q15007: topology; general IHC interpretation).
| Which compartment should guide scoring? | Use nuclear staining as the primary readout; WTAP also has a reported cytoplasmic fraction (UniProt Q15007: subcellular location). A chromogenic slide need not resolve nuclear speckles to support a nuclear call (general IHC interpretation). |
| How much tissue-to-tissue variation is expected? | Expression is described as ubiquitous, yet HPA reports variable nuclear levels: high in several listed populations, low in salivary-gland glandular cells, liver cholangiocytes, skeletal-muscle myocytes, and splenic white-pulp cells, and not detected in cardiomyocytes (UniProt Q15007: tissue specificity; HPA: tissue IHC). |
| How certain is the antibody-based tissue pattern? | The listed HPA010550 antibody has Supported IHC status, and the tissue profile has medium consistency with RNA expression (HPA: antibody validation and tissue reliability). Use the reported tissue levels as reference patterns while reviewing controls and cellular morphology (general IHC practice). |
| What should IF/ICC show? | Expect WTAP enrichment in nuclear speckles; HPA lists this as an enhanced main location and reports ICC-IF images in A-431, U-251MG, and U2OS cells (HPA: subcellular and antibody validation). This is an IF/ICC localisation reference, not an IHC-P protocol or an IHC tissue-intensity standard. |
| Could isoforms change interpretation? | UniProt lists two WTAP isoforms, but the supplied record gives no antibody epitope or isoform-specific staining evidence (UniProt Q15007: isoforms; HPA: supplied antibody record). Do not assign a different cellular pattern to either isoform from these data. |
| Situation | Likely cause | Next action |
|---|---|---|
| High-staining reference tissue shows no nuclear chromogen. | The run may have a detection or staining-step failure (general IHC practice); HPA reports high WTAP in colon glandular cells (HPA: tissue IHC). | Check the positive control, primary-antibody application, retrieval step, detection reagents, and counterstain; document each condition before changing it (general IHC practice). |
| Colour is diffuse across tissue and obscures nuclei. | Nonspecific antibody binding, endogenous detection activity, or excess chromogen development can raise background (general IHC practice). | Compare a no-primary control; review blocking, primary-antibody concentration, washes, and development time against the validated workflow (general IHC practice). |
| Membranes appear more strongly stained than nuclei. | The distribution conflicts with mainly nuclear WTAP and its absence of a transmembrane segment (UniProt Q15007: location and topology). | Recheck cell boundaries and counterstained nuclei; compare controls and review primary-antibody specificity and detection background before scoring (general IHC practice). |
| Cardiomyocytes appear clearly positive. | This differs from the reported not-detected cardiomyocyte pattern (HPA: tissue IHC); cross-reactivity or endogenous detection activity is possible (general IHC practice). | Inspect a no-primary control and a known high-staining tissue on the same run; describe the discrepancy without treating the HPA result as an absolute absence claim (general IHC practice; HPA: Supported, medium consistency). |
| A low-staining population appears negative. | HPA reports low staining in salivary-gland glandular cells and liver cholangiocytes, so weak signal may be hard to distinguish from background (HPA: tissue IHC; general IHC interpretation). | Use an HPA high-staining cell population as a run control and score only signal distinguishable from the no-primary control (HPA: tissue IHC; general IHC practice). |
| Puncta are apparent in IF/ICC but unclear in chromogenic IHC. | HPA's enhanced nuclear-speckle observation comes from ICC-IF, whereas the IHC tissue profile describes nuclear expression (HPA: subcellular and tissue IHC). | For IHC, assess whether staining is predominantly nuclear in the expected cells; reserve a speckle-level localisation judgment for the separate IF/ICC guide (HPA: tissue IHC and subcellular location; general IHC interpretation). |
Experiment evidence
IHC & IF images showing WTAP staining
A04296-1 · IHCImmunohistochemistry (IHC) analyzes of WTAP (H351) pAb in paraffin-embedded human tonsil cancer tissue at 1:50.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000146457-WTAP/tissue/cerebral+cortex#imid_2970948
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000146457-WTAP/tissue/colon#imid_2975914
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000146457-WTAP/tissue/liver#imid_2975923
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000146457-WTAP/tissue/kidney#imid_2975916
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA010550; green = WTAP, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA010550; green = WTAP, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for WTAP IHC & IF
🧪Run bone marrow first: hematopoietic cells should show WTAP staining (HPA: High in bone marrow hematopoietic cells). Run heart muscle as the negative tissue because WTAP is not detected in cardiomyocytes (HPA: Not detected in heart muscle cardiomyocytes); cells without nuclear staining on the bone marrow slide should remain at background, but they are not validated target-negative cells.
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Heart muscle (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show WTAP in A-431, U-251MG, U2OS, U2OS, siRNA 2 (10x), U2OS, siRNA 2 (40x), U2OS, scrambled (10x), U2OS, scrambled (40x), with annotated localisation: Nuclear speckles (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; an isotype control matched to the primary antibody’s host species and clonality; and WTAP knockout tissue or a validated peptide-block control. Quench endogenous peroxidase for chromogenic IHC, especially in peroxidase-rich marrow cells (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected paraffin-section caption does not state its fixative (selected-SKU tissue-IHC caption). Retrieval dependence is unreported; optimize antigen retrieval on the positive tissue alongside controls (standard IHC practice). The supplied evidence does not establish that frozen sections or IF/ICC are easier, although IF/ICC offers a nuclear-speckle localization reference (HPA: nuclear speckles); endogenous peroxidase in marrow can complicate chromogenic scoring (standard IHC practice).
HPA tissue IHC evidence for WTAP
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced WTAP IHC Tips
Troubleshoot WTAP staining in paraffin sections by checking retrieval, nuclear localisation, controls, and scoring before interpreting biological differences.
What retrieval should I use if WTAP nuclear staining is weak?
Start with Tris-EDTA at pH 9.0, heated to 95–98 °C for 20 min (page retrieval rule: nuclear antigen). After cooling, compare nuclear signal and background on sections processed together, keeping the catalog antibody concentration and chromogenic development time constant (standard IHC practice). If staining remains weak, test a shorter or longer heating interval on adjacent sections before trying an alternative buffer, because retrieval can change both signal and morphology (standard IHC practice). Assess success in intact nuclei: WTAP is mainly nuclear, with a smaller cytoplasmic fraction, and localises to nuclear speckles (UniProt Q15007; HPA subcellular).
Can fixation explain weak WTAP staining in my paraffin sections?
The selected A04296-1 image describes paraffin-embedded human tonsil cancer stained at 1:50, but its caption does not state the fixative (A04296-1 IHC caption). WTAP-specific sensitivity to fixation therefore remains unknown from the supplied evidence; do not assign a weak result to overfixation on that basis (A04296-1 IHC caption). For a controlled comparison, record fixation conditions for each specimen, stain matched sections in one run, and keep retrieval, antibody incubation, and DAB development consistent (standard IHC practice). Check whether nuclear staining and tissue morphology vary together before changing the fixation workflow, and document any change against a matched control (standard IHC practice).
Should diffuse cytoplasmic staining count as WTAP positivity?
Prioritise nuclear staining when reading WTAP IHC: tissue staining is described as ubiquitously nuclear at variable levels (HPA tissue IHC), and nuclear speckles are its main subcellular location (HPA subcellular). Some cytoplasmic WTAP is reported, so cytoplasmic signal alone deserves separate recording rather than automatic rejection or acceptance (UniProt Q15007 subcellular). Compare adjacent intact cells and a matched control at the same antibody dilution and DAB development time, noting whether nuclear detail is preserved (standard IHC practice). If strong cytoplasmic staining appears alongside diffuse background or damaged morphology, optimise blocking and detection before interpreting a biological shift (standard IHC practice).
Could an unrecognised WTAP isoform explain inconsistent staining?
WTAP has 2 annotated isoforms, but the supplied record does not map this antibody’s epitope to either isoform (UniProt Q15007 isoforms; A04296-1 IHC caption). Its annotated chain spans residues 1–396, with no signal peptide or transmembrane segment, so routine section permeabilisation does not need to expose a membrane-facing domain (UniProt Q15007 processing and topology). Report isoform-dependent recognition as unresolved until epitope information or an isoform-specific control becomes available (UniProt Q15007 isoforms; A04296-1 IHC caption). When comparing specimens, keep retrieval and staining conditions matched; altered nuclear signal alone cannot identify which isoform is present (standard IHC practice; UniProt Q15007 isoforms).
How can IF help check whether the IHC pattern is cellular and nuclear?
For a separate IF check, multiplex WTAP with a validated marker for the cell type being assessed; glandular cells in colon are one documented high-staining reference (HPA tissue IHC; standard IF practice). Add a nuclear counterstain and inspect whether WTAP concentrates in nuclear puncta, consistent with the reported speckle localisation (HPA subcellular; standard IF practice). Choose a red or far-red WTAP fluorophore when tissue autofluorescence obscures shorter wavelengths, and include single-label controls to check channel bleed-through (standard IF practice). WTAP has no transmembrane segment, so use mild permeabilisation to reach its intracellular epitope while preserving nuclear structure; optimise detergent exposure on matched samples (UniProt Q15007 topology and subcellular; standard IF practice).
What should I change when WTAP DAB staining is widespread?
First separate nuclear signal from diffuse deposit: WTAP is predominantly nuclear, while tissue expression varies across cell populations (UniProt Q15007 subcellular; HPA tissue IHC). Run a no-primary control, block endogenous peroxidase, and review DAB development time to identify detection-related colour (standard chromogenic IHC practice). If the control is clean, titrate the primary antibody and adjust wash stringency on adjacent sections while holding retrieval constant; the selected image used 1:50 in paraffin-embedded tonsil cancer (standard IHC practice; A04296-1 IHC caption). Examine tissue folds, cut edges, and necrotic areas separately from intact nuclei before calling widespread staining WTAP positive (standard IHC practice).
How should I score heterogeneous WTAP nuclear staining? ⚠ ANSWER MARKED FOR VERIFICATION
Define the analysed cell population and score the proportion of positive nuclei plus staining intensity, using an H-score if intensity categories are reproducible (standard IHC practice). Report % positive nuclei and, where cell abundance changes, positive-cell density per mm² of viable analysed tissue (standard IHC practice). Normalise each measure to the counted cells or measured viable area, and apply one staining threshold across slides processed in the same run (standard IHC practice). Record cytoplasmic staining separately because WTAP is mainly nuclear with some cytoplasmic fraction; variable nuclear levels across tissues make a single unqualified “positive” label less informative (UniProt Q15007 subcellular; HPA tissue IHC).
How do I distinguish convincing WTAP positivity from artefact?
A convincing result shows staining in intact nuclei, compatible with the reported ubiquitous nuclear tissue profile and nuclear speckle localisation (HPA tissue IHC; HPA subcellular). Use the expected cell population as context: glandular cells in colon are reported high, whereas cardiomyocytes in heart muscle are reported not detected (HPA tissue IHC). Treat staining restricted to section edges, folds, necrotic tissue, or a no-primary control as suspect, and check endogenous peroxidase blocking if DAB appears without primary antibody (standard chromogenic IHC practice). HPA rates the tissue staining evidence Supported with medium agreement between staining and RNA data, so interpret unexpected patterns alongside morphology and controls (HPA tissue IHC).
Boster reagents
Best WTAP / Pre-mRNA-splicing regulator WTAP IHC Antibodies
A04296-1 has real IHC data from paraffin-embedded human tonsil cancer tissue (IHC image caption). Catalog reactivity lists Human, Mouse, and Rat (catalog: reactivity).
Real IHC data
Anti-WTAP (H351) Antibody
Cat # A04296-1
A04296-1 is listed for IHC in Human, Mouse, and Rat (catalog: applications and reactivity). Its IHC image shows paraffin-embedded human tonsil cancer tissue at 1:50 (IHC image caption).
Which to pick: Choose A04296-1 for paraffin-section IHC; its image documents human tonsil cancer tissue, but does not report the fixative (IHC image caption). No IF/ICC application or image is listed for A04296-1, so the payload does not support an IF/ICC recommendation (catalog: applications and IF images). A04296-1 lists Mouse and Rat reactivity alongside Human, but its supplied tissue image documents Human only (catalog: reactivity; IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q15007 (FL2D_HUMAN, Pre-mRNA-splicing regulator WTAP).
- Human Protein Atlas. WTAP tissue IHC expression (reliability: Supported).
- Human Protein Atlas. WTAP subcellular location (ICC-IF): Localized to the nuclear speckles..
- Human Protein Atlas. WTAP antibody validation summary (1 antibodies).
- Relationship between the genetic expression of WTAP and bladder cancer and patient prognosis. Oncology letters 2018 — PMC6256415.
- WTAP Is Correlated With Unfavorable Prognosis, Tumor Cell Proliferation, and Immune Infiltration in Hepatocellular Carcinoma. Frontiers in oncology 2022 — PMC9035869.
- WTAP is a prognostic marker of high-grade serous ovarian cancer and regulates the progression of ovarian cancer cells. OncoTargets and therapy 2019 — PMC6689666.
- WTAP regulates the production of reactive oxygen species, promotes malignant progression, and is closely related to the tumor microenvironment in glioblastoma. Aging 2024 — PMC11006471.
- PubMed PMID:12444081 — UniProt-cited evidence.
- PubMed PMID:7788527 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for WTAP (Pre-mRNA-splicing regulator WTAP). Expected localisation Nuclear staining in tissue sections (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A04296-1 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Heart muscle. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human tonsil cancer tissue; fixative not specified (datasheet A04296-1); 2. Fixation: Image fixative and duration unreported (datasheet A04296-1); 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 Tris-EDTA buffer, pH 9.0 , 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-WTAP, 1:50-1:200 (datasheet A04296-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Cardiomyocytes may show no detectable staining (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage is unclear (UniProt); Regulation: Ubiquitous expression; variable tissue levels (UniProt; HPA tissue IHC). Evidence: UniProt Q15007, HPA tissue IHC · Supported, HPA ICC-IF · nuclear speckles., Datasheet A04296-1, PMC6689666, PMC11006471. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/WTAP/immunohistochemistry-ihc-guide.