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YWHAQ / 14-3-3 protein theta · IHC design guide
Design Immunohistochemistry for YWHAQ
Plan YWHAQ paraffin-section IHC around mainly cytoplasmic staining in most tissues (HPA tissue IHC). Interpret staining with the HPA antibody’s reported multi-gene reactivity in mind (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
YWHAQ 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 | Mainly cytoplasmic staining in most tissues (HPA tissue IHC) | |
| Staining pattern | Mainly cytoplasmic staining across most tissues (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M03904) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Adipose tissue+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 | HPA staining may include proteins from other genes (HPA tissue IHC) | |
| Regulation | Upregulated in sporadic ALS lumbar spinal cord (UniProt) | |
| Isoform / epitope | No isoforms annotated; one 1–245 chain (UniProt) |
Section 1
Recommended YWHAQ IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet M03904) with a published YWHAQ tissue microarray protocol (PMC6616235).
| Sample | Paraffin-embedded human pancreas cancer tissue; fixative not specified (datasheet M03904) |
| Fixation | Image fixative and duration unreported (datasheet M03904); 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 M03904); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M03904) |
| Primary antibody | Rabbit monoclonal (clone HHF-25) anti-YWHAQ, 1:50 (datasheet M03904) |
| Primary incubation | Overnight at 4 °C (datasheet M03904) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet M03904) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | YWHAQ-positive staining in glandular cells of adrenal gland (HPA tissue IHC: Medium). HPA tissue profile: Mainly cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval: the catalog datasheet specifies it (datasheet M03904), and the published protocol uses the same buffer (PMC6616235).
Section 2
What Is the Expected YWHAQ Staining Pattern?
YWHAQ is a cytoplasmic protein with no transmembrane segment (UniProt P27348). In paraffin sections, expect mainly cytoplasmic staining, including in glandular cells of colon and duodenum and neuronal cells of the caudate (HPA tissue IHC: Medium). Interpret these patterns cautiously: HPA rates the tissue IHC evidence Approved but warns that the antibody targets protein from more than one gene and awaits external verification (HPA tissue IHC).
| Cytoplasmic staining in colon glandular cells. | This fits the reported compartment and a Medium staining cell population (HPA tissue IHC). It supports an expected pattern, but staining alone cannot establish YWHAQ specificity because of HPA's cross-target warning (HPA tissue IHC). |
| Predominantly nuclear or membrane-edge staining. | A dominant nuclear or membrane pattern conflicts with the mainly cytoplasmic tissue pattern (HPA tissue IHC) and the lack of a transmembrane segment (UniProt P27348). Review morphology, counterstain, and controls before assigning it to YWHAQ (general IHC practice). |
| Strong staining in adipocytes or cardiomyocytes. | HPA reports Not detected in these cell populations (HPA tissue IHC). Investigate cross-reactivity, endogenous detection activity, and tissue-specific background with appropriate controls (general IHC practice); an unexpected stain is not proof of another antigen. |
| Diffuse color across cells and surrounding tissue. | A widespread deposit obscures the expected cellular cytoplasm (HPA tissue IHC). Check no-primary and detection controls, reagent blocking, and chromogen development to distinguish background from specific signal (general IHC practice). |
| No signal in colon glandular cells. | Colon glandular cells are reported at Medium level (HPA tissue IHC). First check section quality, retrieval, antibody dilution, and detection controls (general IHC practice); HPA's cross-target warning prevents treating its stain as definitive YWHAQ ground truth (HPA tissue IHC). |
💡Expected YWHAQ appearanceCall a result consistent with the reference pattern when glandular or neuronal cells show chiefly cytoplasmic, approximately Medium staining (HPA tissue IHC); dominant nuclear, membrane-edge, or cell-free color is suspect (HPA tissue IHC; UniProt P27348; general IHC practice).
| Compartment and architecture | Cytoplasm is the annotated location, and no transmembrane segment is recorded (UniProt P27348); score cellular cytoplasm rather than membrane outlines. |
| Tissue selection | HPA reports Medium staining in colon glandular and caudate neuronal cells, but Not detected in cardiomyocytes (HPA tissue IHC). UniProt reports abundant heart expression (UniProt P27348); preserve this discrepancy when selecting controls. |
| Specificity of the reference pattern | HPA calls tissue IHC Approved while warning of protein targets from more than one gene and pending external verification (HPA tissue IHC). Treat the reported pattern as a benchmark requiring independent specificity controls. |
| Protein forms | UniProt records one chain, residues 1–245, no signal peptide or propeptide, and no annotated isoforms (UniProt P27348). These annotations provide no basis to predict a separate shed or isoform-specific IHC pattern. |
| IF/ICC Q: What localisation is expected? | A: Mainly cytosol is supported; nucleoplasm and nucleoli-rim localisation is uncertain (HPA subcellular ICC-IF). Interpret nuclear fluorescence cautiously; IF/ICC requires its own guide. |
| Situation | Likely cause | Next action |
|---|---|---|
| Colon glandular cells stain weakly or not at all. | Possible retrieval, dilution, or detection failure (general IHC practice); HPA reports Medium staining there (HPA tissue IHC). | Check section morphology and controls, then review the antibody's validated IHC-P conditions and detection steps (general IHC practice). |
| Most nuclei are strongly positive. | This differs from mainly cytoplasmic tissue staining (HPA tissue IHC); ICC-IF nuclear locations are uncertain (HPA subcellular ICC-IF). | Compare cytoplasm and nuclei against counterstain and controls; avoid calling nuclear signal a confirmed tissue pattern (general IHC practice; HPA tissue IHC). |
| Adipocytes or muscle cells show prominent color. | These populations are listed as Not detected (HPA tissue IHC); nonspecific binding or endogenous detection activity may contribute (general IHC practice). | Run no-primary and detection controls, then compare the suspicious cells with expected cytoplasmic positives (general IHC practice; HPA tissue IHC). |
| Color coats the section without clear cell boundaries. | Excess background or overdeveloped chromogen can obscure localisation (general IHC practice). | Review blocking, washes, detection controls, and development time; score only interpretable cellular staining (general IHC practice). |
| Heart muscle stains differently from the expression summary. | UniProt describes abundant heart expression, whereas HPA reports cardiomyocytes Not detected by tissue IHC (UniProt P27348; HPA tissue IHC). | Record the discordance and avoid using cardiomyocytes alone as a pass-or-fail control (UniProt P27348; HPA tissue IHC). |
| The expected pattern appears, but identity remains uncertain. | The HPA tissue antibody targets protein from more than one gene, with external verification pending (HPA tissue IHC). | Use an independent specificity control when assigning the signal to YWHAQ; report the antibody and control outcome (general IHC practice; HPA tissue IHC). |
Experiment evidence
IHC & IF images showing YWHAQ staining
M03904 · IHCIHC analysis of YWHAQ using anti-YWHAQ antibody (M03904). YWHAQ was detected in a paraffin-embedded section of human pancreas cancer tissue.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000134308-YWHAQ/tissue/cerebral+cortex#imid_2874420
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000134308-YWHAQ/tissue/colon#imid_2874362
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000134308-YWHAQ/tissue/liver#imid_2874363
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000134308-YWHAQ/tissue/kidney#imid_2874356
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB010286; green = YWHAQ, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB010286; green = YWHAQ, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for YWHAQ IHC & IF
🧪Run cerebellum first: Purkinje cells should show YWHAQ staining (HPA: Medium in Purkinje cells). Run adipose tissue as the negative comparator (HPA: Not detected in adipocytes); on the positive slide, cells judged negative should show counterstain without specific cytoplasmic DAB signal (UniProt P27348: cytoplasm).
Positive control tissue: Adrenal gland (Glandular cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show YWHAQ in A-431, U-251MG, U2OS, A-549, HEK293, KOLF2.1J, with annotated localisation: Cytosol (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and matched rabbit IgG isotype controls (catalog antibody caption: rabbit anti-YWHAQ), plus YWHAQ knockout tissue or cells as a biological negative. Quench endogenous peroxidase for DAB detection and assess cerebellar pigment against the control sections (catalog antibody caption: peroxidase/DAB; HPA: cerebellum).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected M03904 paraffin-section caption does not state a fixative. The caption demonstrates heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required (M03904 tissue-IHC caption). Whether frozen sections or IF are easier is unreported; for IF/ICC, assess the supported cytosolic signal against controls and check tissue autofluorescence (HPA subcellular: cytosol supported).
HPA tissue IHC evidence for YWHAQ
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Caution, targets protein from more than one gene. Pending external verification.). 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 → |
| Caudate | Neuronal cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | 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 YWHAQ IHC Tips
Troubleshoot YWHAQ chromogenic staining in paraffin sections by checking retrieval, compartment, cell type and assay controls together.
How should I adjust retrieval when YWHAQ staining is weak in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 (datasheet M03904). The selected paraffin-section example used this retrieval before overnight incubation with antibody at 1:50 and 4°C (caption M03904). If staining remains weak, compare a modestly longer heating interval on adjacent sections while holding the primary dilution and detection conditions constant (standard IHC practice). Check tissue preservation and a positive control section before attributing absent signal to retrieval, since excessive heating can damage morphology and complicate scoring (standard IHC practice). Record retrieval time, cooling time and section loss alongside staining intensity for each trial (standard IHC practice).
Could fixation explain variable YWHAQ staining between paraffin blocks?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not report a fixative (caption M03904). Compare blocks with documented fixation conditions, using adjacent sections and the same EDTA pH 8.0 retrieval and 1:50 primary incubation used in the selected example (datasheet M03904; caption M03904). Include a consistently processed positive control with each run and assess morphology before comparing signal intensity (standard IHC practice). If fixation histories differ, treat a negative section as inconclusive until processing effects have been assessed, rather than assigning the difference to YWHAQ expression (standard IHC practice). Do not infer fixation tolerance from tissue staining patterns (standard IHC practice).
Where should convincing YWHAQ staining appear in chromogenic IHC?
Expect predominantly cytoplasmic staining when evaluating tissue sections (UniProt P27348 subcellular location; HPA tissue IHC). The HPA subcellular record supports cytosol as the main location, while nucleoplasm and nucleoli-rim signals are uncertain (HPA subcellular). Score cytoplasmic DAB separately from any nuclear staining, using a matched hematoxylin counterstain to identify cell boundaries and nuclei (standard IHC practice). In neuronal tissue, a process-associated pattern can be plausible because YWHAQ is transported axonally to nerve terminals (UniProt P27348 subcellular location). If nuclear staining dominates, review controls and morphology before treating it as target localisation, and document the compartment explicitly (HPA subcellular; standard IHC practice).
How can I assess whether the antibody reports YWHAQ specifically?
The record lists 0 annotated isoforms and no transmembrane segment for YWHAQ (UniProt P27348 isoforms and topology). Those annotations do not establish which epitope the catalog antibody recognizes or whether it distinguishes related proteins (standard IHC interpretation). HPA rates its tissue antibody Approved but cautions that it targets protein from more than one gene and awaits external verification (HPA tissue IHC). For chromogenic sections, compare the catalog antibody with an independently validated YWHAQ reagent or an appropriate depletion control, and require concordant cellular patterns (standard IHC practice). Interpret an isolated DAB-positive compartment conservatively until antibody specificity has been checked (standard IHC practice).
How should I cross-check a chromogenic YWHAQ pattern with IF?
Use IF on matched material as a localisation cross-check, while retaining the paraffin-section IHC result as the primary readout (standard IHC/IF practice). Multiplex YWHAQ with a validated marker for the cell population being scored, and include single-label controls to check channel bleed-through (standard IF practice). Choose a fluorophore channel after inspecting unstained tissue for autofluorescence, which can obscure a weak signal (standard IF practice). Because YWHAQ is primarily cytosolic and has no transmembrane segment, use a controlled permeabilisation trial to provide access to intracellular antigen; do not assume an IHC retrieval condition specifies an IF detergent condition (UniProt P27348 topology; HPA subcellular; standard IF practice).
What should I change if DAB appears across the entire section?
First inspect a no-primary control and tissue edges to separate detection background from cellular staining (standard IHC practice). The selected example used 10% goat serum blocking, a 1:50 rabbit primary overnight at 4°C, and peroxidase-based DAB detection (caption M03904). Apply an endogenous peroxidase block as a general chromogenic IHC step, then check whether DAB develops in the no-primary control (standard IHC practice). If that control is clean, titrate the primary and shorten DAB development while keeping retrieval at EDTA pH 8.0 for the comparison (datasheet M03904; standard IHC practice). Record whether residual signal follows intact cell cytoplasm or damaged regions (standard IHC practice).
How should I quantify YWHAQ IHC across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region of interest before scoring, then measure cytoplasmic staining rather than combining it with uncertain nuclear signal (HPA tissue IHC; HPA subcellular; standard IHC practice). An H-score can combine the percentage of cells at intensity grades 0–3; also report the percentage positive and the number of evaluable cells (standard IHC practice). For spatial comparisons, report positive-cell density per mm² of viable tissue or the relevant compartment (standard IHC practice). Normalise comparisons to the same cell type, viable area, batch controls and image-analysis thresholds, and exclude folds and necrosis consistently (standard IHC practice).
When is a YWHAQ-positive DAB signal likely to be misleading?
A credible signal should follow intact cell cytoplasm, the main tissue pattern reported for YWHAQ (HPA tissue IHC; UniProt P27348 subcellular location). Treat staining restricted to section edges, folds or necrotic areas as suspect, and inspect a no-primary control for endogenous enzyme or detection background (standard IHC practice). Check cell identity: HPA reports medium staining in several glandular and neuronal cell populations, while its tissue antibody carries a cross-gene caution (HPA tissue IHC). Strongly nuclear-only staining deserves separate review because HPA calls its additional nuclear locations uncertain (HPA subcellular). Reconcile unexpected patterns with morphology, matched controls and an independent specificity check before calling them biological changes (standard IHC practice).
Boster reagents
Best YWHAQ / 14-3-3 protein theta IHC Antibodies
M03904 has a real IHC image from a human pancreas cancer paraffin section; IHC, IF and ICC are listed for human, mouse and rat (M03904 IHC caption; catalog applications/reactivity).
Real IHC data
Anti-14-3-3 Theta YWHAQ Rabbit Monoclonal Antibody
Cat # M03904
M03904 will render with its own IHC image from a paraffin-embedded human pancreas cancer section (M03904 IHC caption). IF and ICC are listed applications, but no IF image is supplied; human, mouse and rat are listed as reactive species (catalog applications/reactivity; catalog IF image list).
Which to pick: Choose M03904 for paraffin-section chromogenic IHC: its image caption documents EDTA pH 8 retrieval, 1:50 primary antibody and HRP/DAB detection; the fixative is unreported (M03904 IHC caption). For IF/ICC, M03904 is listed for both, with an IF dilution of 1:50, but has no supplied IF image (catalog applications/IF dilution/IF image list). For human, mouse or rat samples, M03904 is the listed rabbit monoclonal option; the supplied tissue image documents human tissue only (catalog host/clone/reactivity; M03904 IHC caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P27348 (1433T_HUMAN, 14-3-3 protein theta).
- Human Protein Atlas. YWHAQ tissue IHC expression (reliability: Approved).
- Human Protein Atlas. YWHAQ subcellular location (ICC-IF): Mainly localized to the cytosol. In addition localized to the nucleoplasm and nucleoli rim..
- Human Protein Atlas. YWHAQ antibody validation summary (3 antibodies).
- Regulatory factor X5 promotes hepatocellular carcinoma progression by transactivating tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein theta and suppressing apoptosis. Chinese medical journal 2019 — PMC6616235.
- PubMed PMID:2015305 — UniProt-cited evidence.
- PubMed PMID:8515476 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for YWHAQ (14-3-3 protein theta). Expected localisation Mainly cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M03904). Validated IHC antibody: M03904 at 1:50 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human pancreas cancer tissue; fixative not specified (datasheet M03904); 2. Fixation: Image fixative and duration unreported (datasheet M03904); 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 M03904); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M03904); 8. Primary antibody: Rabbit monoclonal (clone HHF-25) anti-YWHAQ, 1:50 (datasheet M03904); 9. Primary incubation: Overnight at 4 °C (datasheet M03904). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: HPA staining may include proteins from other genes (HPA tissue IHC); Isoform / epitope: No isoforms annotated; one 1–245 chain (UniProt); Regulation: Upregulated in sporadic ALS lumbar spinal cord (UniProt). Evidence: UniProt P27348, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the cytosol. In addition localized to the…, Datasheet M03904, PMC6616235. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/YWHAQ/immunohistochemistry-ihc-guide.