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DDAH2 / Putative hydrolase DDAH2 · IHC design guide
Design Immunohistochemistry for DDAH2
Plan chromogenic DDAH2 IHC on paraffin sections using the catalog antibody (datasheet PB10001). Assess cytoplasmic staining across tissues, but interpret intensity cautiously because antibody staining has low consistency with RNA expression (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
DDAH2 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 | Glandular cells show cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PB10001) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining has low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | No expression regulator annotated (UniProt) | |
| Isoform / epitope | No annotated isoforms or cleaved chains (UniProt) |
Section 1
Recommended DDAH2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet PB10001) is accompanied by four published DDAH2 IHC protocols (PMC4778151; PMC7689360; PMC4912010; PMC3818215).
| Sample | Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet PB10001) |
| Fixation | Image fixative and duration unreported (datasheet PB10001); 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 PB10001); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB10001) |
| Primary antibody | Rabbit anti-DDAH2, 0.5-1μg/ml (datasheet PB10001) |
| Primary incubation | Overnight at 4 °C (datasheet PB10001) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB10001) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | DDAH2-positive staining in glandular cells of adrenal gland (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet PB10001). The published protocols use their stated conditions (PMC4778151; PMC7689360).
Section 2
What Is the Expected DDAH2 Staining Pattern?
DDAH2 is a non-transmembrane protein reported in the cytoplasm and mitochondria (UniProt O95865: topology and localization). In paraffin-section IHC, expect cytoplasmic staining in selected glandular, respiratory epithelial and hematopoietic cells (HPA: tissue IHC). Treat tissue patterns as provisional: HPA rates its tissue IHC evidence Uncertain because antibody staining has low consistency with RNA expression (HPA: reliability).
| Cytoplasmic staining in adrenal or appendix glandular cells, or bone marrow hematopoietic cells (HPA: Medium). | This fits the reported IHC pattern and offers a comparison for the specimen. Medium is HPA’s staining category, not a calibrated intensity target for every run (HPA: tissue IHC). |
| Predominantly nuclear or sharply cell-surface staining, with little cytoplasmic signal. | That compartment does not fit reported cytoplasmic tissue staining or UniProt’s cytoplasmic and mitochondrial localization (HPA: tissue IHC; UniProt O95865: localization). Recheck morphology and antibody specificity before scoring it as DDAH2. |
| Strong staining concentrated in adipocytes, skeletal myocytes or vaginal squamous epithelial cells. | HPA reports Low staining in these cells (HPA: tissue IHC). A conspicuously stronger pattern warrants comparison with a Medium-staining tissue and checks for cross-reactivity or endogenous chromogenic activity (HPA: tissue IHC; standard IHC practice). |
| Diffuse color across cells, extracellular spaces and the section background. | A widespread deposit without a discernible cytoplasmic cell pattern is difficult to assign to DDAH2 (HPA: tissue IHC). Review blocking, washes and the detection-only control for nonspecific or endogenous signal (standard IHC practice). |
| No staining in adrenal or appendix glandular cells tested as a positive control. | HPA reports Medium staining in these cells, so a blank control tissue makes a specimen-negative call unreliable (HPA: tissue IHC). Check tissue integrity, retrieval, antibody application and detection with run controls (standard IHC practice). |
💡Expected DDAH2 appearanceA plausible positive is discernible cytoplasmic staining in HPA-reported Medium cells, such as adrenal glandular cells; dominant nuclear, surface or uniform background color is suspect (HPA: tissue IHC; UniProt O95865: localization).
| Which compartment should guide IHC scoring? | Use the cytoplasmic pattern documented across most HPA tissues; UniProt also lists mitochondria, but chromogenic tissue IHC alone need not resolve individual mitochondria (HPA: tissue IHC; UniProt O95865: localization; standard IHC practice). |
| How strong is the tissue-pattern evidence? | HPA labels tissue IHC Uncertain and reports low staining-versus-RNA consistency. Its listed Medium and Low cells are useful comparisons, not definitive positive or negative classifications (HPA: reliability and tissue IHC). |
| What do topology and processing imply? | No transmembrane segment, signal peptide, propeptide or annotated processed chain supports a required surface or secreted pattern; UniProt lists a single chain, residues 1–285 (UniProt O95865: topology and processing). |
| IF/ICC Q: What localization is expected? | A: HPA supports mitochondrial localization in ICC-IF images from EFO-21, SK-MEL-30 and U2OS; UniProt also lists cytoplasm and reports mitochondrial translocation after IL1B stimulation in chondrocytes (HPA: subcellular; UniProt O95865: localization). This does not establish an IF/ICC protocol or change the tissue IHC reliability rating (HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Positive-control tissue is blank. | An assay or tissue problem is possible when HPA-listed Medium cells also lack staining; the HPA rating remains Uncertain (HPA: tissue IHC and reliability). | Inspect morphology and run controls; verify the intended antigen retrieval, antibody application and chromogenic detection steps for the validated IHC procedure (standard IHC practice). |
| Only nuclei or cell borders are colored. | The dominant compartment conflicts with HPA’s cytoplasmic IHC profile and UniProt’s cytoplasmic and mitochondrial localization (HPA: tissue IHC; UniProt O95865: localization). | Compare with the control tissue, inspect counterstain and boundaries, and check a detection-only control before accepting the signal (standard IHC practice). |
| The whole section has a diffuse chromogenic haze. | Diffuse color can reflect nonspecific detection or endogenous enzyme activity rather than a readable cell pattern (standard IHC practice; HPA: cytoplasmic IHC profile). | Check detection-only controls, blocking and wash steps; score cells only where signal can be separated from section background (standard IHC practice). |
| Low-category cells appear as strongly stained as the control. | HPA lists adipocytes, skeletal myocytes and vaginal squamous epithelial cells as Low, but its tissue evidence is Uncertain (HPA: tissue IHC and reliability). | Compare sections stained in the same run and review cell identity and background controls; describe the discrepancy without declaring those cells DDAH2-negative (standard IHC practice; HPA: tissue IHC). |
| A brain section shows staining despite low UniProt tissue detection. | UniProt reports very low brain levels, while HPA lists Medium staining in selected neuronal or endothelial cells; these observations require cell-level interpretation (UniProt O95865: tissue specificity; HPA: tissue IHC). | Record the stained cell population and compartment, then compare with controls; avoid treating whole-brain abundance as a cell-specific IHC exclusion (HPA: tissue IHC; standard IHC practice). |
| Mitochondrial ICC-IF and diffuse cytoplasmic IHC look different. | HPA supports mitochondrial ICC-IF localization but describes cytoplasmic staining in most tissues; the methods resolve localization differently (HPA: subcellular and tissue IHC; standard microscopy practice). | Interpret each result against its own control and imaging resolution; do not require paraffin-section chromogenic IHC to show discrete mitochondrial puncta (standard microscopy practice). |
Experiment evidence
IHC & IF images showing DDAH2 staining
PB10001 · IHCIHC analysis of DDAH2 using anti-DDAH2 antibody (PB10001). DDAH2 was detected in a paraffin-embedded section of human lung cancer tissue. He
PB10001 · IHCIHC analysis of DDAH2 using anti-DDAH2 antibody (PB10001). DDAH2 was detected in a paraffin-embedded section of human placenta tissue. Heat
PB10001 · IFIF analysis of DDAH2 using anti-DDAH2 antibody (PB10001). DDAH2 was detected in immunocytochemical section of U20S cells. Enzyme antigen ret
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000213722-DDAH2/tissue/cerebral+cortex#imid_3890936
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000213722-DDAH2/tissue/colon#imid_3890862
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000213722-DDAH2/tissue/liver#imid_3890871
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000213722-DDAH2/tissue/kidney#imid_3890864
HPA · ICC-IF EFO-21Human Protein Atlas immunofluorescence, EFO-21, antibody HPA012389; green = DDAH2, blue = nucleus, red = microtubules.
HPA · ICC-IF EFO-21Human Protein Atlas immunofluorescence, EFO-21, antibody HPA012389; green = DDAH2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for DDAH2 IHC & IF
🧪Run bronchus first and score staining in respiratory epithelial cells, which HPA rates Medium (HPA: bronchus, respiratory epithelial cells, Medium). HPA detects DDAH2 in all 45 scored tissues, so there is no HPA negative tissue; use no-primary and isotype controls, and expect cells without specific staining on the positive slide to match their local control background rather than treating them as proven DDAH2-negative (HPA: no negative rows; detected in all 45 scored tissues).
Positive control tissue: Adrenal gland (Glandular cells, HPA Medium)
Negative control tissue: None in HPA: DDAH2 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show DDAH2 in EFO-21, SK-MEL-30, U2OS, with annotated localisation: Mitochondria (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide; an isotype control using nonimmune rabbit IgG matched to the primary antibody’s class, clonality, and concentration; and, if available, a DDAH2 knockout specimen as a biological negative (caption: rabbit primary and biotinylated goat anti-rabbit secondary). In bronchus, block endogenous peroxidase and check for background from endogenous biotin before interpreting DAB staining (HPA: bronchus respiratory epithelial cells; caption: streptavidin–biotin detection with DAB).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the fixative in the selected PB10001 paraffin-section caption is unreported (caption: fixative not stated). Heat retrieval with EDTA at pH 8.0 was used in that IHC example, but retrieval dependence is unreported (caption: EDTA retrieval, pH 8.0). Whether frozen sections or IF are easier is unreported; ICC-IF images support mitochondrial localization, while endogenous biotin can complicate the caption’s streptavidin–biotin detection method (HPA: mitochondria, supported; caption: streptavidin–biotin detection).
HPA tissue IHC evidence for DDAH2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Low consistency between antibody staining and RNA expression data. 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 → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: DDAH2 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced DDAH2 IHC Tips
Troubleshoot DDAH2 staining in paraffin sections by checking retrieval, controls, cell location, and scoring against the available IHC evidence.
How should I optimize retrieval when DDAH2 staining is weak?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet PB10001). The selected image used this condition on human lung cancer tissue, followed by 10% goat serum and 1 μg/ml primary antibody overnight at 4°C (caption PB10001). If staining remains weak, compare shorter and longer heating times on adjacent sections while holding antibody concentration and detection constant (general IHC practice). Score tissue preservation alongside cytoplasmic signal, since the reported tissue pattern is broadly cytoplasmic and its staining reliability is uncertain (HPA tissue IHC). Record retrieval and cooling times consistently across runs (general IHC practice).
Could fixation be masking DDAH2 in paraffin sections?
Target-specific fixation sensitivity is unknown because the selected paraffin-section caption does not state a fixative (caption PB10001). Record the actual fixative and fixation duration for each specimen, then compare matched sections using EDTA pH 8.0 retrieval and 1 μg/ml antibody (datasheet PB10001; caption PB10001; general IHC practice). Inspect tissue preservation and cytoplasmic signal together, using a primary-antibody omission control to identify detection background (HPA tissue IHC; general IHC practice). Do not infer a DDAH2-specific fixation effect from its lack of annotated transmembrane segments, glycosylation sites, or modified residues (UniProt O95865 topology and annotations).
Should DDAH2 appear diffuse or punctate in tissue sections?
Expect cytoplasmic staining as the primary tissue pattern, while allowing mitochondrial enrichment within cells (HPA tissue IHC; UniProt O95865 subcellular location). Mitochondrial localization is supported in cell imaging, and cytosol-to-mitochondrion movement after IL1B stimulation is reported in chondrocytes (HPA subcellular; UniProt O95865). In chromogenic sections, use cell morphology and a serial-section mitochondrial marker to assess punctate signal before assigning DAB granules to organelles (general IHC practice). Compare a 1 μg/ml primary condition with an omission control, and record predominantly nuclear or membranous staining as discordant with reported localization (caption PB10001; UniProt O95865; general IHC practice).
Can isoforms or processing explain inconsistent DDAH2 staining?
The supplied record lists 0 isoforms, no signal peptide or propeptide, and a single 1–285 chain (UniProt O95865). It also lists 0 glycosylation sites and 0 modified residues, so those annotations offer no explanation for different staining patterns (UniProt O95865). The antibody epitope is not provided; obtain its mapped sequence, if available, and compare staining after EDTA pH 8.0 retrieval in paired sections (datasheet PB10001; general IHC practice). If specimens differ, compare fixation records, retrieval runs, and omission controls before assigning the discrepancy to a DDAH2 molecular form (caption PB10001; general IHC practice).
How can I verify DDAH2 localization by multiplex IF?
Pair DDAH2 with a marker for the expected cell type, such as a respiratory epithelial marker when examining bronchus (HPA tissue IHC; general IF practice). Choose a spectrally separated fluorophore after checking tissue autofluorescence in an unstained section, and include single-label controls for channel bleed-through (general IF practice). Permeabilize cells to give the antibody access to cytoplasmic and mitochondrial epitopes; DDAH2 has no annotated transmembrane segment, and both locations are reported (UniProt O95865 topology and location; HPA subcellular). Assess diffuse cytoplasmic signal and mitochondrial overlap without assuming the IHC caption’s 1 μg/ml condition transfers to IF (caption PB10001; HPA tissue IHC; HPA subcellular; general IF practice).
What should I check when DAB obscures DDAH2 staining?
First inspect a primary-antibody omission section for detection background, including endogenous peroxidase or biotin-associated signal (general IHC practice). The selected workflow used a biotinylated goat anti-rabbit secondary, streptavidin-biotin detection, and DAB after 10% goat serum blocking (caption PB10001). Include a peroxidase block and compare adjacent sections under identical DAB development conditions (general IHC practice). If diffuse background persists, reassess blocking, secondary specificity, washing, and development time while keeping the reported 1 μg/ml primary condition as a reference (caption PB10001; general IHC practice). Judge cytoplasmic staining against morphology and controls (HPA tissue IHC; general IHC practice).
How should I score heterogeneous DDAH2 staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell compartment and region before scoring, because the reported tissue pattern is cytoplasmic and staining reliability is uncertain (HPA tissue IHC). Report the percentage of positive cells and intensity using an H-score, or count positive cells per mm² when cell density varies (general IHC practice). Normalize counts to viable tissue area or the number of eligible cells, excluding necrosis and damaged edges by the same rule across sections (general IHC practice). Set one positivity threshold using omission controls and apply it consistently to sections stained at the reported 1 μg/ml primary condition (caption PB10001; general IHC practice). Score distinct cell populations separately when morphology permits (general IHC practice).
How do I distinguish true DDAH2 staining from artefact?
A credible signal follows cytoplasmic or mitochondrial localization in intact cells, with tissue interpretation anchored to the reported cytoplasmic pattern (UniProt O95865; HPA tissue IHC; HPA subcellular). Check the stained cell type using morphology and a companion marker, then examine omission controls for endogenous peroxidase or biotin-associated DAB signal (caption PB10001; general IHC practice). Flag nuclear-only staining, cut edges, necrosis, and diffuse extracellular deposits as possible artefacts before assigning them to DDAH2 (UniProt O95865 location; general IHC practice). HPA rates tissue staining reliability uncertain because antibody staining and RNA expression have low consistency, so confirm claims with an independent antibody or orthogonal assay (HPA tissue IHC; general IHC practice).
Boster reagents
Best DDAH2 / Putative hydrolase DDAH2 IHC Antibodies
PB10001 has IHC images from human paraffin sections and IF data from U20S cells (catalog image captions); listed reactivity includes human, mouse and rat (catalog reactivity).
Real IHC data
Anti-DDAH2 Antibody ®
Cat # PB10001
PB10001 is listed for IHC, IF and ICC, with human, mouse and rat reactivity (catalog applications and reactivity). Its IHC captions show human lung cancer and placenta paraffin sections; its IF caption shows U20S cells (catalog image captions).
Which to pick: Choose PB10001 for chromogenic IHC of paraffin sections: its human tissue captions report EDTA retrieval and 1 μg/ml primary antibody, but do not report the fixative (PB10001 IHC image captions). For IF/ICC, PB10001 is listed for both applications and has a U20S cell IF image at 2 μg/ml (catalog applications; PB10001 IF image caption). PB10001 also lists mouse and rat reactivity, although its supplied IHC and IF images do not demonstrate those species (catalog reactivity; PB10001 image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O95865 (DDAH2_HUMAN, Putative hydrolase DDAH2).
- Human Protein Atlas. DDAH2 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. DDAH2 subcellular location (ICC-IF): Localized to the mitochondria..
- Human Protein Atlas. DDAH2 antibody validation summary (2 antibodies).
- Dimethylarginine dimethylaminohydrolase 2 promotes tumor angiogenesis in lung adenocarcinoma. Virchows Archiv : an international journal of pathology 2016 — PMC4778151.
- Upregulation of DDAH2 Limits Pulmonary Hypertension and Right Ventricular Hypertrophy During Chronic Hypoxia in Ddah1 Knockout Mice. Frontiers in physiology 2020 — PMC7689360.
- Dimethylarginine dimethylaminohydrolase-2 deficiency promotes vascular regeneration and attenuates pathological angiogenesis. Experimental eye research 2016 — PMC4912010.
- Expressed sequence tags for bovine muscle satellite cells, myotube formed-cells and adipocyte-like cells. PloS one 2013 — PMC3818215.
- PubMed PMID:10493931 — UniProt-cited evidence.
- PubMed PMID:11042152 — UniProt-cited evidence.
- PubMed PMID:10384126 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for DDAH2 (Putative hydrolase DDAH2). Expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PB10001). Validated IHC antibody: PB10001 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet PB10001); 2. Fixation: Image fixative and duration unreported (datasheet PB10001); 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 PB10001); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB10001); 8. Primary antibody: Rabbit anti-DDAH2, 0.5-1μg/ml (datasheet PB10001); 9. Primary incubation: Overnight at 4 °C (datasheet PB10001). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining has low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: No annotated isoforms or cleaved chains (UniProt); Regulation: No expression regulator annotated (UniProt). Evidence: UniProt O95865, HPA tissue IHC · Uncertain, HPA ICC-IF · mitochondria., Datasheet PB10001, PMC4778151, PMC7689360. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/DDAH2/immunohistochemistry-ihc-guide.