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DAPK1 / Death-associated protein kinase 1 · IHC design guide
Design Immunohistochemistry for DAPK1
Plan DAPK1 staining in paraffin sections using the catalog antibody at 2 μg/ml with chromogenic detection (datasheet A01161-1). Esophageal squamous cells provide an HPA high staining reference, while adipocytes provide an HPA undetected comparison (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
DAPK1 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 staining in most tissues (HPA tissue IHC) | |
| Staining pattern | High cytoplasmic signal in esophageal squamous cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A01161-1) | |
| Positive control | Esophagus+4 more · see all | |
| Negative control | Adipose tissue+3 more · see all |
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 A01161-1) | |
| Caveat | Antibody staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Isoform 2 occurs in intestine and CRC (UniProt) | |
| Isoform / epitope | 4 isoforms; no extracellular domain; verify epitope coverage (UniProt) |
Section 1
Recommended DAPK1 IHC & IF Protocols
The catalog antibody protocol uses EDTA pH 8.0 retrieval (datasheet A01161-1). Published DAPK1 IHC protocols below cover liver cancer TMA and placental sections (PMC5520959; PMC5429897).
| Sample | Paraffin-embedded human colorectal adenocarcinoma tissue; fixative not specified (datasheet A01161-1) |
| Fixation | Image fixative and duration unreported (datasheet A01161-1); 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 A01161-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A01161-1) |
| Primary antibody | Rabbit anti-DAPK1, 2-5 μg/ml (datasheet A01161-1) |
| Primary incubation | Overnight at 4 °C (datasheet A01161-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A01161-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | DAPK1-positive staining in squamous epithelial cells of esophagus (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 HIER for the catalog antibody (datasheet A01161-1); assess the published retrieval conditions when adapting either workflow (PMC5520959; PMC5429897).
Section 2
What Is the Expected DAPK1 Staining Pattern?
DAPK1 is expected mainly in the cytoplasm, with cytoskeletal association; it has no transmembrane segment (UniProt P53355). In paraffin-section IHC, look for cytoplasmic staining in the cell types reported by HPA, including squamous epithelial cells and keratinocytes (HPA tissue IHC). HPA describes cytoplasmic expression in most tissues, but its tissue IHC reliability is Approved with medium consistency between antibody staining and RNA data (HPA tissue IHC).
| Cytoplasmic staining in esophageal or tonsillar squamous epithelial cells, or skin keratinocytes. | This matches HPA's reported high staining in those cells and its overall cytoplasmic tissue pattern (HPA tissue IHC). Compare cells within the section; a brown deposit alone does not establish that the stained cell is a reported positive cell type (standard IHC interpretation). |
| Predominantly nuclear staining, with little or no cytoplasmic staining. | A nuclear-only pattern does not match the cytoplasmic IHC profile (HPA tissue IHC; UniProt P53355). Review morphology, detection background and controls before calling it DAPK1; HPA's centrosome finding comes from ICC-IF and does not make nuclear-only IHC an expected result (HPA subcellular ICC-IF). |
| Strong staining in adipocytes, smooth muscle cells or hippocampal glial cells. | HPA reports DAPK1 as not detected in those specified cells (HPA tissue IHC). Check that the stained cells were identified correctly, then investigate cross-reactivity or detection activity. Treat discordance as a reason to validate the result, since HPA's tissue IHC and RNA data have medium consistency (HPA tissue IHC). |
| Diffuse brown haze across cells and surrounding tissue, without a clear cytoplasmic boundary. | A field-wide haze obscures the cell-based cytoplasmic pattern reported by HPA (HPA tissue IHC). Uneven blocking, excessive antibody or detection background are general IHC possibilities, not established DAPK1-specific effects (standard IHC practice). Interpret expression only after appropriate controls distinguish specific signal from background. |
| No detectable signal in a section containing a reported high-staining cell population. | Absence of staining in esophageal squamous epithelial cells, for example, conflicts with HPA's reported high level there (HPA tissue IHC). First verify that those cells are present and preserved; then assess the antibody, detection reagents and run controls (standard IHC practice). One failed section cannot establish biological absence. |
💡Expected DAPK1 appearanceCall a positive IHC result when staining is predominantly cytoplasmic in a reported high-staining population, such as skin keratinocytes, and is clearly stronger than controlled background (HPA tissue IHC); isolated nuclear-only signal or similarly strong staining in HPA not-detected cell populations warrants investigation (HPA tissue IHC; UniProt P53355).
| Choice of comparison tissue and cell population | HPA reports high staining in esophageal and tonsillar squamous epithelium, nasopharyngeal respiratory epithelium, placental trophoblasts, skeletal myocytes, skin keratinocytes and vaginal squamous epithelium (HPA tissue IHC). Its not-detected calls are cell-specific: adipocytes, hippocampal glia, parathyroid glandular cells and smooth muscle cells (HPA tissue IHC). Score the named cells, not an entire tissue as uniformly positive or negative. |
| Strength of the HPA evidence | The tissue IHC profile is Approved, with medium consistency against RNA expression data (HPA tissue IHC). HPA lists IHC Approved status for HPA040472, HPA048436 and CAB037302; HPA003516 has no listed IHC status in the supplied antibody record (HPA antibodies). These statuses support comparison with the reported pattern but do not prove specificity in every section. |
| Isoforms and epitope coverage | UniProt lists four DAPK1 isoforms and reports isoform 2 in normal intestinal tissue and colorectal carcinomas (UniProt P53355). The supplied record gives no antibody epitope or isoform coverage. An unexpected intestinal result therefore cannot be assigned to an isoform from this evidence alone; check the chosen antibody's stated epitope before making an isoform-specific interpretation. |
| Topology and processing | UniProt reports no transmembrane segment, signal peptide or propeptide, and identifies one DAPK1 chain spanning residues 1–1430 (UniProt P53355). This supports a cellular, mainly cytoplasmic interpretation alongside the tissue IHC profile (HPA tissue IHC). It supplies no basis for predicting staining in a membrane compartment or a shed extracellular product. |
| Q: What should IF/ICC show? | A: HPA reports the centrosome as the approved main ICC-IF location and lists images from A-549, HaCaT, U2OS, A-431 and U-251MG cells (HPA subcellular ICC-IF). UniProt also reports cytoplasmic and cytoskeletal localization (UniProt P53355). Interpret this as an application-specific observation; it does not replace the cytoplasmic tissue IHC expectation (HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Reported high-staining cells are present, but the section has no signal. | A failed staining run or unsuitable antibody conditions are possible (standard IHC practice); HPA reports high staining in several defined cell populations (HPA tissue IHC). | Confirm the cell identity, then check a known-positive section in the same run, antibody handling and detection reagents (standard IHC practice). If controls fail, repeat the run before scoring DAPK1 absent. |
| Expected cytoplasmic staining is weak or hard to separate from counterstain. | Suboptimal staining or detection settings may reduce contrast (standard IHC practice). HPA reports a cytoplasmic tissue profile but supplies no target-specific fixation or retrieval effect (HPA tissue IHC). | Compare the positive control and background together. Review the chosen antibody's IHC-P instructions for dilution and antigen retrieval, and adjust one general workflow variable at a time (standard IHC practice). Do not infer a DAPK1-specific retrieval requirement from HPA staining levels. |
| The strongest signal is nuclear rather than cytoplasmic. | The compartment disagrees with UniProt's cytoplasmic localization and HPA's tissue IHC profile (UniProt P53355; HPA tissue IHC). Nonspecific staining or scoring error remains possible (standard IHC interpretation). | Recheck the nucleus against the counterstain, inspect positive and negative controls, and confirm whether cytoplasm is also stained. Withhold a positive DAPK1 call if the pattern remains nuclear-only. |
| Adipocytes or smooth muscle cells stain as strongly as the proposed positive cells. | Those specified cells are reported as not detected (HPA tissue IHC). Cell misidentification, cross-reactivity or detection background could explain the mismatch (standard IHC interpretation). | Verify cell identity on the section and compare with reported high-staining cells processed in the same run (HPA tissue IHC). Check negative detection controls; investigate persistent discordance before using those cells as evidence of DAPK1 expression. |
| Brown staining spreads across the section, including areas without a clear cellular pattern. | Diffuse detection background can mask specific cytoplasmic staining (standard IHC practice); it does not resemble HPA's described cell-based profile (HPA tissue IHC). | Inspect reagent-only controls, blocking and wash steps, and the detection system (standard IHC practice). Reduce background and reassess staining within identifiable cells before comparing intensity across tissues. |
| A low-staining cell population is scored as equivalent to a high-staining one. | HPA reports low staining in cerebral cortex endothelial cells and rectal glandular cells, while skin keratinocytes are high (HPA tissue IHC). Section-to-section intensity alone can mislead without matched processing and controls (standard IHC interpretation). | Compare the named cell populations using consistent staining and imaging conditions, record cytoplasmic signal relative to local background, and keep HPA's medium IHC–RNA consistency in view (HPA tissue IHC). Avoid converting an intensity difference into an absolute expression claim. |
Experiment evidence
IHC & IF images showing DAPK1 staining
A01161-1 · IHCIHC analysis of DAPK1 using anti-DAPK1 antibody (A01161-1). DAPK1 was detected in a paraffin-embedded section of human colorectal adenocarci
A01161-1 · IHCIHC analysis of DAPK1 using anti-DAPK1 antibody (A01161-1). DAPK1 was detected in a paraffin-embedded section of human liver cancer tissue.
M01161 · IHCImmunohistochemical analysis of paraffin-embedded human breast cancer, using DAP Kinase 1 Antibody.
M01161 · IFImmunofluorescent analysis of Hela cells, using DAP Kinase 1 Antibody .
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000196730-DAPK1/tissue/cerebral+cortex#imid_15413339
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000196730-DAPK1/tissue/colon#imid_15413265
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000196730-DAPK1/tissue/liver#imid_15413258
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000196730-DAPK1/tissue/kidney#imid_15413267
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA003516; green = DAPK1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA003516; green = DAPK1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for DAPK1 IHC & IF
🧪Run skin first: keratinocytes should stain strongly (HPA: High in skin keratinocytes). Run adipose tissue as the negative comparator (HPA: Not detected in adipocytes); any nonkeratinocyte cells used as internal negative references on the skin slide should show only background, with their status confirmed rather than assumed from the keratinocyte result.
Positive control tissue: Esophagus (Squamous epithelial cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show DAPK1 in A-549, HaCaT, U2OS, A-431, U-251MG, with annotated localisation: Centrosome (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a concentration-matched rabbit isotype control (catalog caption: rabbit primary antibody); use DAPK1 knockout material, if available, as a biological negative. Quench endogenous peroxidase for DAB detection (catalog caption: peroxidase secondary and DAB), and distinguish brown skin pigment from chromogen in the controls (skin: standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A01161-1 paraffin-section caption does not state the fixative (selected-SKU caption). The caption reports heat retrieval in EDTA at pH 8.0 before IHC, but does not establish that retrieval is required (selected-SKU caption). No matched comparison establishes whether frozen sections or IF are easier; skin pigment can complicate chromogenic scoring (skin: standard IHC practice), while HPA reports mainly centrosomal ICC-IF localization and UniProt reports cytoplasmic and cytoskeletal localization (HPA: subcellular; UniProt P53355: subcellular).
HPA tissue IHC evidence for DAPK1
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 |
|---|---|---|---|---|
| Esophagus | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Nasopharynx | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Placenta | Trophoblastic cells | High | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | High | Protein (IHC) | HPA → |
| Skin | Keratinocytes | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced DAPK1 IHC Tips
Troubleshoot DAPK1 staining in paraffin sections by checking retrieval, cell type, subcellular pattern, and controls before comparing staining intensity.
Which retrieval condition should I try first for DAPK1 in paraffin sections?
Start with heat-mediated retrieval in EDTA pH 8.0 (datasheet A01161-1). The catalog antibody detected DAPK1 in a paraffin-embedded human colorectal adenocarcinoma section after that retrieval, followed by 2 μg/ml primary antibody overnight at 4°C (datasheet A01161-1). If staining is weak, first check heating consistency, section adhesion, and whether the primary incubation matches that tested condition (standard IHC practice; datasheet A01161-1). Compare any fallback retrieval condition on serial sections with a positive control, because altered heating can change background as well as signal (standard IHC practice). Do not infer a required retrieval condition from DAPK1 phosphorylation sites (UniProt P53355).
Could fixation explain weak or uneven DAPK1 staining?
The selected tissue image reports a paraffin-embedded section but does not state its fixative, so DAPK1-specific fixation sensitivity is unknown (datasheet A01161-1). Record the actual fixative and fixation history for each specimen before comparing sections (standard IHC practice). On serial sections, hold EDTA pH 8.0 retrieval and the 2 μg/ml primary condition constant while checking whether weak staining follows a particular processing batch (datasheet A01161-1; standard IHC practice). Review morphology and section-wide staining alongside a positive control to identify processing problems (standard IHC practice). HPA tissue patterns and DAPK1 topology or phosphorylation sites do not establish a fixation effect for this antibody (HPA: tissue IHC; UniProt P53355).
What staining compartment is plausible for DAPK1, and when should I question it?
Expect predominantly cytoplasmic staining in tissue sections: HPA reports cytoplasmic expression in most tissues, while UniProt also associates DAPK1 with the cytoskeleton, microtubules, and cortical actin fibers (HPA: tissue IHC; UniProt P53355). HPA reports a mainly centrosomal location in cell imaging, so a small focal signal may warrant comparison with a centrosome marker in a separate IF experiment (HPA: subcellular). DAPK1 has no transmembrane segment; a sharp membrane-only chromogenic pattern deserves specificity checks (UniProt P53355 topology; standard IHC practice). Compare the suspect pattern with a no-primary control and expected cytoplasmic staining on adjacent sections (standard IHC practice). Score the relevant cell population rather than assigning diffuse staining across the whole section to DAPK1 (standard IHC practice).
How can isoforms or epitope accessibility complicate DAPK1 IHC?
DAPK1 has 4 reported isoforms, and isoform 2 is expressed in normal intestinal tissue and colorectal carcinomas (UniProt P53355). The supplied caption documents detection in a paraffin-embedded human colorectal adenocarcinoma section but does not identify the antibody epitope or establish which isoforms it recognizes (datasheet A01161-1). Check the catalog antibody’s epitope information before treating a staining difference as an isoform difference (standard IHC practice). DAPK1 contains a kinase domain at residues 13–275, a Roc domain at 681–955, and a death domain at 1312–1396 (UniProt P53355). Its reported phosphorylation sites likewise do not establish that this antibody is modification-specific (UniProt P53355; datasheet A01161-1).
How should I investigate DAPK1 localisation by IF alongside tissue IHC?
Use IF as a separate localisation check; the selected antibody evidence here documents chromogenic staining of a paraffin section, not an IF protocol for that antibody (datasheet A01161-1). Multiplex DAPK1 with a marker for the cell population under study so that signal can be assigned to cells, and include single-label controls when evaluating overlap (standard IF practice). Choose fluorophores after examining tissue autofluorescence in an unstained section, favoring channels with clearer separation from that background (standard IF practice). DAPK1 has no transmembrane segment and is reported in the cytoplasm and cytoskeleton, so permeabilisation should permit access to intracellular epitopes (UniProt P53355 topology and subcellular location; standard IF practice). Compare any focal signal cautiously with HPA’s mainly centrosomal cell-imaging pattern (HPA: subcellular).
What should I check when DAPK1 chromogenic staining appears diffuse?
First inspect a no-primary section for staining caused by detection reagents, endogenous peroxidase, or the chromogen (standard IHC practice). The documented tissue condition used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, a peroxidase-conjugated secondary for 30 minutes at 37°C, and DAB development (datasheet A01161-1). Match those documented conditions when troubleshooting the catalog antibody, then compare controlled changes in blocking, washing, or primary concentration on serial sections (datasheet A01161-1; standard IHC practice). Check whether pigment, folds, necrotic areas, or section edges account for the apparent signal before scoring it (standard IHC practice). HPA’s broad cytoplasmic tissue profile means widespread staining alone does not prove specificity (HPA: tissue IHC).
How should I quantify DAPK1 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and cytoplasmic compartment before scoring, because HPA describes cytoplasmic expression in most tissues and variable staining among listed cell types (HPA: tissue IHC). For chromogenic IHC, report the percentage of positive cells and intensity, or calculate an H-score from intensity-weighted cell percentages using the same thresholds across sections (standard IHC practice). If counting discrete positive cells, express density per mm² of evaluable tissue and exclude folds or necrotic regions (standard IHC practice). Normalize comparisons to the same cell type, sampled area, staining run, and positive-control performance (standard IHC practice). Keep antibody concentration and retrieval consistent, using the documented 2 μg/ml and EDTA pH 8.0 condition as the reference (datasheet A01161-1).
How can I distinguish credible DAPK1 staining from artefact?
Credible staining should fit the sampled cell population and a predominantly cytoplasmic distribution, while a membrane-only pattern warrants closer review because DAPK1 lacks a transmembrane segment (HPA: tissue IHC; UniProt P53355 topology). HPA reports high staining in skin keratinocytes and no detection in adipose-tissue adipocytes, providing contrasting cell-type observations rather than universal positive and negative controls (HPA: tissue IHC). Discount staining confined to section edges, folds, or necrotic regions, and investigate signal present in a no-primary control as possible detection or endogenous-enzyme background (standard IHC practice). Compare the pattern across serial sections processed with EDTA pH 8.0 retrieval and the documented antibody condition (datasheet A01161-1). Interpret discordant cases cautiously because HPA rates its tissue staining and RNA agreement as medium consistency (HPA: Approved reliability).
Boster reagents
Best DAPK1 / Death-associated protein kinase 1 IHC Antibodies
IHC images show DAPK1 staining in human paraffin sections; an IF image shows staining in HeLa cells (A01161-1 and M01161 image captions). Both antibodies list Human, Mouse, and Rat reactivity (catalog).
Real IHC data
Anti-DAPK1 Antibody ®
Cat # A01161-1
Real IHC data
Anti-DAP Kinase 1 DAPK1 Rabbit Monoclonal Antibody
Cat # M01161
A01161-1 has IHC images from human colorectal adenocarcinoma, liver cancer, and lung cancer paraffin sections (A01161-1 IHC captions). M01161 has an IHC image from a human breast cancer paraffin section and an IF image from HeLa cells (M01161 image captions).
Which to pick: Choose A01161-1 for paraffin-section IHC when a documented starting workflow is useful: its human tissue captions specify EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody; the fixative is unreported (A01161-1 IHC captions). Choose M01161 for IF/ICC because it is a rabbit monoclonal listed for both applications, with a HeLa-cell IF image; its IHC caption supports paraffin sections but does not report the fixative (M01161 catalog; M01161 image captions). For cross-species planning, both SKUs list Human, Mouse, and Rat reactivity, while their pictured IHC examples are human tissue (catalog reactivity; IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P53355 (DAPK1_HUMAN, Death-associated protein kinase 1).
- Human Protein Atlas. DAPK1 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. DAPK1 subcellular location (ICC-IF): Mainly localized to the centrosome..
- Human Protein Atlas. DAPK1 antibody validation summary (4 antibodies).
- Association of Protein Expression and Methylation of DAPK1 with Clinicopathological Features in Invasive Ductal Carcinoma Patients from Kashmir. Asian Pacific journal of cancer prevention : APJCP 2019 — PMC6825784.
- Protection of FK506 against neuronal apoptosis and axonal injury following experimental diffuse axonal injury. Molecular medicine reports 2017 — PMC5428482.
- DAPK1 as an independent prognostic marker in liver cancer. PeerJ 2017 — PMC5520959.
- Placental DAPK1 and autophagy marker LC3B-II are dysregulated by TNF-α in a gestational age-dependent manner. Histochemistry and cell biology 2017 — PMC5429897.
- PubMed PMID:7828849 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:17974005 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for DAPK1 (Death-associated protein kinase 1). Expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A01161-1). Validated IHC antibody: A01161-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Esophagus (Squamous epithelial cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colorectal adenocarcinoma tissue; fixative not specified (datasheet A01161-1); 2. Fixation: Image fixative and duration unreported (datasheet A01161-1); 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 retrieval: EDTA pH 8.0 (datasheet A01161-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A01161-1); 8. Primary antibody: Rabbit anti-DAPK1, 2-5 μg/ml (datasheet A01161-1); 9. Primary incubation: Overnight at 4 °C (datasheet A01161-1). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A01161-1); Caveat: Antibody staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 4 isoforms; no extracellular domain; verify epitope coverage (UniProt); Regulation: Isoform 2 occurs in intestine and CRC (UniProt). Evidence: UniProt P53355, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the centrosome., Datasheet A01161-1, PMC5520959, PMC5429897. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/DAPK1/immunohistochemistry-ihc-guide.