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DAPK3 / Death-associated protein kinase 3 · IHC design guide
Design Immunohistochemistry for DAPK3
Plan DAPK3 paraffin IHC with the catalog antibody at 1:50–1:200 (datasheet). Assess cytoplasmic and membranous staining, using smooth muscle cells as a high-staining reference (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
DAPK3 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic and membranous tissue staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic/membranous in most tissues; distinct in smooth muscle (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Adipose tissue+4 more · see all | |
| Negative control | Liver+2 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); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A03300-2) | |
| Caveat | Thr-299 phosphorylation can alter nuclear/cytoplasmic distribution (UniProt) | |
| Regulation | Widely expressed; no inducer reported (UniProt) | |
| Isoform / epitope | 2 isoforms; confirm epitope coverage for both (UniProt) |
Section 1
Recommended DAPK3 IHC & IF Protocols
Compare the catalog antibody's IHC-P protocol (datasheet) with the published mouse testis IHC workflow (PMC12061289).
| Sample | Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A03300-2) |
| Fixation | Image fixative and duration unreported (datasheet A03300-2); 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-DAPK3, 1:50-1:200 (datasheet A03300-2) |
| 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 | DAPK3-positive staining in adipocytes of adipose tissue (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and membranous expression in most tissues, distinct expression in smooth muscle cells. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA HIER at pH 9.0, 95–98 °C for 20 min (page retrieval); the published workflow does not specify retrieval (PMC12061289).
Section 2
What Is the Expected DAPK3 Staining Pattern?
DAPK3 is predominantly cytoplasmic but can shuttle into the nucleus; it has no transmembrane segment (UniProt O43293: localization, topology). In paraffin-section IHC, expect cytoplasmic and membranous staining across many tissues, with distinct staining in smooth muscle cells (HPA: tissue IHC profile). HPA rates its tissue IHC pattern Approved, with medium consistency between staining and RNA expression data (HPA: tissue IHC reliability).
| Strong cytoplasmic staining in smooth muscle cells, with staining along cell borders (HPA: High in smooth muscle; cytoplasmic and membranous profile). | This fits the reported tissue pattern. High staining in adipocytes, lung macrophages, or rectal glandular cells is also plausible (HPA: High in those cells). Judge each cell type separately within the section (general IHC practice). |
| Staining is confined to extracellular material or luminal deposits, without a cellular pattern (UniProt O43293: intracellular localization). | That distribution does not fit the annotated locations and warrants an artefact check (UniProt O43293: localization; general IHC practice). Nuclear staining alone is not automatically wrong: DAPK3 can shuttle into the nucleus (UniProt O43293: localization). |
| Strong signal appears in liver cholangiocytes or parathyroid glandular cells (HPA: Not detected in those cells). | Treat it as unexpected, then assess antibody cross-reactivity and endogenous detection activity with appropriate controls (HPA: tissue IHC; general IHC practice). An HPA 'Not detected' call is a reference observation, not proof that every specimen must be negative (HPA: tissue IHC). |
| Color spreads across stroma, empty spaces, and many cell types without clear cell boundaries (general IHC practice). | This is more consistent with background than interpretable DAPK3 staining (general IHC practice). Check the no-primary control, blocking, washes, detection reagents, and chromogen development before assigning a tissue pattern (general IHC practice). |
| A smooth muscle control has no detectable cell-associated signal (HPA: High in smooth muscle cells). | The run may have inadequate sensitivity or a failed detection step (general IHC practice). Review section quality, retrieval, antibody dilution, and detection controls before calling a test specimen negative (general IHC practice). |
💡Expected DAPK3 appearanceA convincing positive shows clear cell-associated, chiefly cytoplasmic staining, including high signal in smooth muscle cells; border staining can occur, while signal confined to extracellular deposits is suspect (HPA: tissue IHC profile; UniProt O43293: localization; general IHC practice).
| Cellular compartment (UniProt O43293; HPA: tissue IHC and ICC-IF) | Cytoplasm predominates in UniProt, while HPA tissue IHC reports cytoplasmic and membranous staining. HPA ICC-IF supports nucleoplasm and cytosol; assess nuclear signal in context rather than rejecting it (UniProt O43293: localization; HPA: tissue IHC, ICC-IF). |
| Choice of reference cells (HPA: tissue IHC) | Smooth muscle cells and lung macrophages are High, whereas liver cholangiocytes are Not detected (HPA: tissue IHC). This gives useful contrasting cell populations, although HPA rates overall staining-to-RNA consistency as medium (HPA: reliability). |
| Isoforms and antibody coverage (UniProt O43293; HPA: antibody validation) | UniProt lists 2 isoforms, including both in bladder smooth muscle (UniProt O43293: isoforms, tissue specificity). The supplied antibody record does not establish isoform-specific recognition, so do not interpret staining as an isoform measurement (HPA: HPA028569 validation record). |
| Retrieval and fixation assessment (general IHC practice) | Optimize retrieval against a positive control when establishing paraffin-section IHC (general IHC practice). The supplied UniProt and HPA records give no DAPK3-specific fixation sensitivity or retrieval condition; do not infer one from tissue staining or protein modifications (UniProt O43293; HPA: tissue IHC). |
| Detection background (general chromogenic IHC practice) | Endogenous enzyme activity or nonspecific detection can mimic cell staining; use controls appropriate to the detection chemistry (general IHC practice). Compare signal with the reported positive and negative cell populations before attributing it to DAPK3 (HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Smooth muscle cells stain weakly or remain blank despite their expected High level (HPA: smooth muscle cells). | The section or IHC run may have inadequate sensitivity; HPA staining does not specify which preparation step failed (HPA: tissue IHC; general IHC practice). | Verify the positive-control section and detection reagents, then assess retrieval and antibody dilution under the local IHC procedure (general IHC practice). Do not assign a DAPK3-specific fixation defect without supporting evidence. |
| Liver cholangiocytes stain strongly, contrary to the HPA 'Not detected' observation (HPA: liver cholangiocytes). | Cross-reactivity or endogenous detection activity is possible; the image alone cannot distinguish them (general IHC practice). | Compare a no-primary control and the expected cell pattern, and check the detection chemistry (general IHC practice; HPA: tissue IHC). Treat the discrepancy as unresolved until controls support a specific signal. |
| The slide has diffuse color that obscures cytoplasmic and border staining (HPA: tissue IHC profile). | Excess detection background can prevent compartment scoring (general IHC practice). | Inspect the no-primary control, blocking and washes; adjust antibody dilution or chromogen development if needed (general IHC practice). Score DAPK3 only where individual cells and their staining compartments remain distinguishable. |
| Most cells show nuclear-only staining, with little cytoplasmic signal (UniProt O43293: predominantly cytoplasmic). | Nuclear localization is biologically possible, but an exclusively nuclear pattern across the section differs from the predominant localization and HPA tissue IHC profile (UniProt O43293; HPA: tissue IHC). | Check the positive control, counterstain, and no-primary control; compare cell-by-cell with cytoplasmic staining (general IHC practice). Record reproducible nuclear signal separately rather than automatically scoring it as artefact (UniProt O43293: nuclear localization). |
| Cell-border staining is mistaken for proof that DAPK3 is a membrane-spanning protein (HPA: membranous tissue IHC profile). | Staining appearance does not establish protein topology; UniProt lists no transmembrane segment (UniProt O43293: topology). | Describe the observed border pattern without assigning membrane insertion (HPA: tissue IHC; UniProt O43293: topology). Confirm that the signal is cell-associated and that control sections behave as expected (general IHC practice). |
| IF/ICC question: Can nucleoplasmic signal be expected? | Yes. HPA supports nucleoplasm and cytosol in ICC-IF; UniProt also lists nuclear localization (HPA: subcellular ICC-IF; UniProt O43293: localization). | Interpret IF/ICC on its own guide page. The supplied HPA028569 record lists ICC as Supported and IHC as Approved; these statuses do not provide an IF protocol or establish identical staining across preparations (HPA: antibody validation). |
Experiment evidence
IHC & IF images showing DAPK3 staining
A03300-2 · IHCImmunohistochemistry (IHC) analyzes of ZIP-kinase (K261) pAb in paraffin-embedded human breast carcinoma tissue at 1:100.
A03300-1 · IFImmunofluorescence of ZIPK in Jurkat cells with ZIPK antibody at 10 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000167657-DAPK3/tissue/cerebral+cortex#imid_11184399
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000167657-DAPK3/tissue/colon#imid_11208552
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000167657-DAPK3/tissue/liver#imid_11208533
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000167657-DAPK3/tissue/kidney#imid_11208547
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA028569; green = DAPK3, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA028569; green = DAPK3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for DAPK3 IHC & IF
🧪Run lung first; macrophages should stain (HPA: High in lung macrophages). Use spleen red-pulp cells as the negative comparison (HPA: Not detected in spleen red-pulp cells); on the lung slide, treat only cells that remain at background under the control conditions as internal negatives, since the supplied HPA row does not identify a negative lung cell type.
Positive control tissue: Adipose tissue (Adipocytes, HPA High)
Negative control tissue: Liver (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show DAPK3 in A-431, U-251MG, U2OS, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), KOLF2.1J, with annotated localisation: Nucleoplasm (supported), Cytosol (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide and a host-matched nonimmune IgG control for the polyclonal primary (selected product caption: pAb); use DAPK3 knockout material as a biological negative where available. For lung macrophages, quench endogenous peroxidase and check pigment against the no-primary slide in chromogenic IHC; check autofluorescence if evaluating IF (HPA: High in lung macrophages).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence; the selected SKU caption shows paraffin-section IHC at 1:100 but does not report the fixative (selected product caption: paraffin-embedded breast carcinoma tissue at 1:100; fixative not stated). Antigen-retrieval dependence is unreported, so optimize retrieval on the chosen sections; the supplied evidence does not establish whether frozen sections or IF are easier, although ICC-IF images support nucleoplasmic and cytosolic localization (HPA subcellular: supported nucleoplasm and cytosol). Lung macrophage pigment and endogenous peroxidase can complicate chromogenic interpretation, and autofluorescence can complicate IF interpretation (HPA: High in lung macrophages; standard IHC/IF practice).
HPA tissue IHC evidence for DAPK3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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 |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Adipocytes | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Lung | Macrophages | High | Protein (IHC) | HPA → |
Section 3
Advanced DAPK3 IHC Tips
Troubleshoot DAPK3 staining in paraffin sections by checking retrieval, cell identity, compartment, and controls before interpreting chromogenic signal.
What retrieval conditions should I start with for DAPK3 in paraffin sections?
Start with Tris-EDTA pH 9.0 heat-induced antigen retrieval at 95–98 °C for 20 min (page retrieval setting). Allow sections to cool in retrieval buffer, then compare staining with a section processed without primary antibody to assess detection background (standard IHC practice). If signal remains weak, test another retrieval buffer or pH on adjacent sections while holding antibody concentration and detection conditions constant (standard IHC practice). Score cytoplasmic and nuclear staining separately because DAPK3 can occupy both compartments; a change in their relative visibility alone does not establish improved specificity (UniProt O43293 localization; standard IHC practice).
How should I troubleshoot fixation when DAPK3 staining is weak?
The selected antibody was imaged in paraffin-embedded human breast carcinoma at 1:100, but its caption does not state a fixative (A03300-2 tissue-IHC caption). Target-specific sensitivity to fixation is therefore unknown from the supplied evidence; record each specimen’s fixative and fixation interval before comparing staining (A03300-2 tissue-IHC caption; standard IHC practice). Inspect section integrity and compare similarly processed control sections before changing retrieval or antibody concentration, since multiple preparation steps can affect apparent signal (standard IHC practice). If fixation histories differ, process matched sections together and report that limitation when comparing nuclear and cytoplasmic staining (standard IHC practice; UniProt O43293 localization).
Should DAPK3 staining be nuclear, cytoplasmic, or both?
Evaluate nuclear and cytoplasmic chromogenic signal independently: DAPK3 is predominantly cytoplasmic but can shuttle between cytoplasm and nucleus (UniProt O43293 localization). Supported nucleoplasmic and cytosolic locations in cell imaging make either compartment plausible, but those observations do not validate a particular tissue stain (HPA subcellular; standard IHC practice). Check whether staining follows intact cell boundaries and recurs in the same cell population across adjacent sections; diffuse precipitate over empty spaces is suspect (standard IHC practice). Record any centrosomal, spindle, or midbody pattern as an observation requiring appropriate morphology and controls, rather than assigning those structures from chromogen alone (UniProt O43293 localization; standard IHC practice).
Can this stain distinguish DAPK3 isoforms or phosphorylation states?
Do not assign an isoform from staining unless the antibody’s documented epitope distinguishes it: DAPK3 has 2 reported isoforms (UniProt O43293 isoforms; standard IHC practice). Both isoforms are reported in bladder smooth muscle, so staining there alone cannot identify which isoform contributed the signal (UniProt O43293 tissue specificity; standard IHC practice). DAPK3 has multiple modified residues, including Thr-299; a total-protein stain does not establish phosphorylation at that site without a validated site-specific reagent (UniProt O43293 modified residues; standard IHC practice). Record the antibody epitope, when available, and compare controls processed under identical retrieval and detection conditions before drawing isoform or modification conclusions (standard IHC practice).
How can IF help resolve ambiguous DAPK3 IHC localisation?
Use IF on a separately validated preparation to check whether ambiguous chromogenic signal aligns with intact nuclei or cytoplasm; supported DAPK3 locations include both (HPA subcellular; standard IF practice). Multiplex DAPK3 with a validated marker of the expected cell type, such as smooth muscle cells where distinct tissue expression is reported, and include single-label controls (HPA tissue IHC; standard IF practice). Choose fluorophores after inspecting tissue autofluorescence in an unstained control, favoring channels with better signal separation for the specimen (standard IF practice). Because DAPK3 has no transmembrane segment and has intracellular locations, permeabilise sufficiently to expose the relevant intracellular epitope while checking morphology (UniProt O43293 topology and localization; standard IF practice).
What should I check when DAPK3 chromogenic staining looks diffuse?
Compare a no-primary section with the stained section to locate signal from the detection system or tissue itself (standard IHC practice). Include a peroxidase block in an enzyme-based chromogenic workflow and inspect whether pigment, precipitate, or endogenous enzyme activity persists without primary antibody (standard IHC practice). If background rises across several cell types, titrate the antibody around the caption’s 1:100 condition and keep retrieval, incubation, and imaging consistent between sections (A03300-2 tissue-IHC caption; standard IHC practice). Judge the result against cellular morphology: DAPK3 may appear in cytoplasm and nucleus, whereas uniform deposits crossing tissue boundaries warrant further investigation (UniProt O43293 localization; standard IHC practice).
How should I quantify DAPK3 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because DAPK3 staining can be cytoplasmic or nuclear and tissue expression varies by cell type (UniProt O43293 localization; HPA tissue IHC). For chromogenic sections, report an H-score or the percentage of positive cells within the annotated population, using fixed intensity thresholds across the comparison (standard IHC practice). If counting discrete positive cells, report density per mm² of evaluable tissue and state how excluded necrotic or damaged regions were handled (standard IHC practice). Normalise counts to the relevant cell population or evaluable area, and analyse matched sections with the same staining and imaging settings (standard IHC practice).
When is DAPK3 staining convincing rather than an artefact?
A convincing result follows identifiable cells and an appropriate intracellular compartment: DAPK3 is reported in cytoplasm and nucleus, with distinct expression in smooth muscle cells (UniProt O43293 localization; HPA tissue IHC). Compare expected cell populations with nearby reference populations; HPA reports high staining in lung macrophages but no detection in liver cholangiocytes (HPA tissue IHC). Treat signal restricted to section edges, necrotic zones, or no-primary controls as possible processing or detection artefact, including endogenous enzyme activity (standard IHC practice). Finally, interpret any disagreement cautiously because the HPA tissue profile has Approved reliability with medium consistency between antibody staining and RNA data (HPA tissue IHC).
Boster reagents
Best DAPK3 / Death-associated protein kinase 3 IHC Antibodies
Anti-DAPK3 antibodies have IHC data from paraffin-embedded human breast carcinoma tissue (A03300-2 image caption) and IF/ICC data from Jurkat cells (A03300-1 image captions); both list Human, Mouse and Rat reactivity (catalog).
Real IHC data
Anti-ZIP-kinase (K261) DAPK3 Antibody
Cat # A03300-2
Real IF data
Anti-ZIPK DAPK3 Antibody
Cat # A03300-1
A03300-2 lists IHC and shows staining of paraffin-embedded human breast carcinoma tissue at 1:100 (catalog applications; A03300-2 image caption). A03300-1 lists IF/ICC and shows staining in Jurkat cells at 10 μg/mL (catalog applications; A03300-1 image captions).
Which to pick: Choose A03300-2 for paraffin-section IHC; its image caption supports that processing context, but does not report the fixative (A03300-2 image caption). Choose A03300-1 for IF/ICC based on its application list and Jurkat cell images (catalog applications; A03300-1 image captions). Both list Human, Mouse and Rat reactivity for cross-species planning, while clonality is unreported for both (catalog reactivity and clone fields).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O43293 (DAPK3_HUMAN, Death-associated protein kinase 3).
- Human Protein Atlas. DAPK3 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. DAPK3 subcellular location (ICC-IF): Mainly localized to the nucleoplasm and cytosol. In addition localized to vesicles and primary cilium..
- Human Protein Atlas. DAPK3 antibody validation summary (1 antibodies).
- Activation of AKT negatively regulates the pro-apoptotic function of death-associated protein kinase 3 (DAPK3) in prostate cancer. Cancer letters 2016 — PMC4884664.
- An Inhibitor of Death-Associated Protein Kinase 3 (DAPK3) Disrupts Hippo Signaling and Intestinal Epithelial Regeneration in Murine DSS-Induced Colitis. Digestive diseases and sciences 2026 — PMC12969504.
- DAPK3 is Essential for DBP-Induced Autophagy of Mouse Leydig Cells. Advanced science (Weinheim, Baden-Wurttemberg, Germany) 2025 — PMC12061289.
- PubMed PMID:9488481 — UniProt-cited evidence.
- PubMed PMID:10356987 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for DAPK3 (Death-associated protein kinase 3). Expected localisation Cytoplasmic and membranous tissue staining (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A03300-2 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Adipose tissue (Adipocytes); negative Liver. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A03300-2); 2. Fixation: Image fixative and duration unreported (datasheet A03300-2); 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-DAPK3, 1:50-1:200 (datasheet A03300-2); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (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 A03300-2); Caveat: Thr-299 phosphorylation can alter nuclear/cytoplasmic distribution (UniProt); Isoform / epitope: 2 isoforms; confirm epitope coverage for both (UniProt); Regulation: Widely expressed; no inducer reported (UniProt). Evidence: UniProt O43293, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm and cytosol. In addition…, Datasheet A03300-2, PMC12061289. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/DAPK3/immunohistochemistry-ihc-guide.