DDAH1 / N(G),N(G)-dimethylarginine dimethylaminohydrolase 1 · IHC design guide

Design Immunohistochemistry for DDAH1

Plan chromogenic DDAH1 IHC in paraffin sections using kidney proximal tubules as a high-staining reference (HPA tissue IHC). Compare cytoplasmic staining across cell types and keep fixation consistent across sections (HPA tissue IHC; standard IHC practice).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for DDAH1 (IHC for DDAH1): expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC), antibody PB10000, validated IHC image, and IHC protocol steps — Printable **DDAH1** IHC protocol sheet — expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC), antibody PB10000, controls and protocol steps. Open the full DDAH1 IHC guide →

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DDAH1 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	Cytoplasmic; high in kidney proximal tubule cell bodies (HPA tissue IHC)
	Antigen retrieval	Citrate pH 6 HIER, heat-mediated (datasheet PB10000)
	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	Staining and RNA have medium consistency (HPA tissue IHC)
	Regulation	Expression regulation is unreported (UniProt)
	Isoform / epitope	2 isoforms; epitope coverage is unspecified (UniProt)

Section 1

Recommended DDAH1 IHC & IF Protocols

The catalog antibody protocol uses citrate pH 6 retrieval (datasheet: PB10000). Published DDAH1 IHC protocols cover eye, xenograft, penile and gastric tissue (PMC4912010; PMC12376576; PMC5245815; PMC5579338).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded rat brain tissues; fixative not specified (datasheet PB10000)
Fixation	Image fixative and duration unreported (datasheet PB10000); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat retrieval: Citrate pH 6, 20 min (datasheet PB10000)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet PB10000)
Primary antibody	Rabbit anti-DDAH1, 0.5-1μg/ml (datasheet PB10000)
Primary incubation	Overnight at 4 °C (datasheet PB10000)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB10000)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	DDAH1-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues, including kidney. No signal in the no-primary control.

💡Decision noteStart with heat-mediated citrate pH 6 retrieval for the catalog antibody (datasheet: PB10000). The xenograft report mentions retrieval but gives no conditions (PMC12376576).

Section 2

What Is the Expected DDAH1 Staining Pattern?

In paraffin-section IHC, expect mainly cytoplasmic DDAH1 staining, with strong signal in kidney proximal tubule cell bodies and several other cell populations (HPA: tissue IHC). HPA rates the tissue profile Enhanced, while reporting medium consistency between staining and RNA expression (HPA: tissue IHC reliability). DDAH1 has no annotated transmembrane segment; UniProt does not assign it a subcellular location (UniProt O94760: topology and subcellular annotation).

What am I looking at on my slide?
Strong cytoplasmic staining in kidney proximal tubule cell bodies.	This matches a high-signal reference population (HPA: High in kidney proximal tubules). Compare staining in the identified cells with surrounding tissue; a uniformly dark section is harder to interpret as a cell-specific result (general IHC practice).
Predominantly nuclear, sharply membranous, or extracellular staining in an IHC section.	Check the pattern against HPA's mainly cytoplasmic tissue profile before calling it DDAH1 (HPA: tissue IHC profile). HPA also reports plasma membrane, cytosol, and additional nucleolar localization in ICC/IF, so compartment differences require assay-specific interpretation (HPA: subcellular ICC/IF).
Strong staining in adipocytes or lymph-node germinal center cells.	These cell populations are listed as not detected in HPA tissue IHC (HPA: adipocytes and germinal center cells). Recheck cell identification and controls; apparent signal may reflect nonspecific binding or endogenous chromogen-generating activity (general IHC practice).
Diffuse brown haze across cells and extracellular areas.	A diffuse pattern does not resolve HPA's reported cell-specific cytoplasmic distribution (HPA: tissue IHC profile). In chromogenic IHC, inadequate blocking, excess detection reagent, or residual endogenous activity can create background; examine control sections before scoring (general IHC practice).
No signal in kidney proximal tubules.	An absent result conflicts with the high staining reported for proximal tubule cell bodies (HPA: High in kidney proximal tubules). Check tissue preservation, primary antibody, retrieval, and detection controls before interpreting the specimen as negative (general IHC practice).

💡Expected DDAH1 appearanceCall the IHC result positive when proximal tubule cell bodies show high, mainly cytoplasmic staining (HPA: kidney proximal tubules; HPA: tissue IHC profile); diffuse haze or dominant staining in HPA-listed negative cells warrants a control review (HPA: tissue IHC; general IHC practice).

How each factor affects the staining
Tissue and cell selection	Kidney proximal tubules provide a high-staining reference, while adipocytes are reported as not detected (HPA: kidney proximal tubules; HPA: adipocytes). Use the named cell populations when comparing sections, since a whole-tissue average can obscure their different patterns (general IHC practice).
Strength of the reference pattern	HPA labels its tissue profile Enhanced but describes medium consistency between antibody staining and RNA expression (HPA: tissue IHC reliability). 2 listed antibodies have Enhanced IHC status (HPA: HPA006308 and HPA071064); these ratings support the reference pattern without guaranteeing every specimen.
Compartment and topology	The tissue profile is mainly cytoplasmic (HPA: tissue IHC), and UniProt annotates no transmembrane segment or subcellular location (UniProt O94760: topology and subcellular annotation). Neither record establishes a mandatory membrane pattern for chromogenic paraffin-section IHC.
Isoform coverage	UniProt lists 2 DDAH1 isoforms (UniProt O94760: isoforms). The supplied evidence does not map either antibody's epitope to an isoform, so staining intensity cannot be assigned to a particular isoform from these records.
What does ICC/IF add?	HPA reports approved cytosol and plasma-membrane localization, with additional approved nucleolar localization in ICC/IF (HPA: subcellular ICC/IF). Use that finding to interpret an IF image in its own assay context; the paraffin-section IHC reference remains mainly cytoplasmic (HPA: tissue IHC).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Kidney proximal tubules are unstained.	The expected high-signal reference is absent (HPA: High in kidney proximal tubules); a failed retrieval or detection step is one possible technical cause (general IHC practice).	Confirm the proximal tubules are present, then review the IHC-validated antibody's recommended retrieval and dilution conditions and run a positive control through the same staining batch (general IHC practice).
All tissue structures have similar brown staining.	Widespread chromogen can arise from endogenous enzyme activity or detection background (general IHC practice), obscuring HPA's cell-specific cytoplasmic profile (HPA: tissue IHC).	Review a no-primary control, the appropriate endogenous-activity block, wash steps, and detection reagent concentration before scoring any cell population (general IHC practice).
Adipocytes stain as strongly as the positive reference.	HPA reports adipocytes as not detected (HPA: adipocytes). Strong signal there raises concern about cell identification, nonspecific binding, or background; HPA's category alone does not identify the cause.	Verify tissue morphology and compare the no-primary control with the same staining run. Reassess primary antibody dilution and blocking if the unexpected signal persists (general IHC practice).
Only nuclei appear positive in paraffin-section IHC.	A nuclear-dominant IHC pattern differs from HPA's mainly cytoplasmic tissue profile (HPA: tissue IHC). Additional nucleolar localization is an ICC/IF observation and does not by itself validate this IHC result (HPA: subcellular ICC/IF).	Check counterstain and chromogen separation, then compare positive and no-primary controls and repeat the assay under the validated IHC conditions if needed (general IHC practice).
Weak staining is seen in pancreas but strong staining in kidney.	HPA lists pancreatic exocrine glandular cells as Low and kidney proximal tubules as High (HPA: pancreas; HPA: kidney). Different intensities across those named cell types can therefore be consistent with the reference profile.	Score each identified cell population against its own HPA category and inspect staining controls before increasing detection sensitivity (HPA: tissue IHC; general IHC practice).
The IF image appears more membranous than the IHC section.	Plasma-membrane and cytosol localization are approved in HPA ICC/IF, while HPA describes tissue IHC as mainly cytoplasmic (HPA: subcellular ICC/IF; HPA: tissue IHC).	Interpret each image against its assay-specific HPA reference. Keep the paraffin-section IHC call anchored to identifiable cells and cytoplasmic staining (HPA: tissue IHC; general IHC practice).

Source: UniProt O94760 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing DDAH1 staining

IHC analysis of DDAH1 using anti-DDAH1 antibody (PB10000). DDAH1 was detected in paraffin-embedded section of rat brain tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-DDAH1 Antibody (PB10000) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

PB10000 · IHCIHC analysis of DDAH1 using anti-DDAH1 antibody (PB10000). DDAH1 was detected in paraffin-embedded section of rat brain tissues. Heat mediat

IHC analysis of DDAH1 using anti-DDAH1 antibody (PB10000). DDAH1 was detected in paraffin-embedded section of mouse brain tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-DDAH1 Antibody (PB10000) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

PB10000 · IHCIHC analysis of DDAH1 using anti-DDAH1 antibody (PB10000). DDAH1 was detected in paraffin-embedded section of mouse brain tissues. Heat medi

IF analysis of DDAH1 using anti-DDAH1 antibody (PB10000). DDAH1 was detected in immunocytochemical section of U20S cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2μg/mL rabbit anti-DDAH1 Antibody (PB10000) overnight at 4°C. DyLight®550 Conjugated Goat Anti-Rabbit IgG (BA1135) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

PB10000 · IFIF analysis of DDAH1 using anti-DDAH1 antibody (PB10000). DDAH1 was detected in immunocytochemical section of U20S cells. Enzyme antigen ret

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000153904-DDAH1/tissue/cerebral+cortex#imid_21033435

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000153904-DDAH1/tissue/colon#imid_21034233

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000153904-DDAH1/tissue/liver#imid_21034234

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000153904-DDAH1/tissue/kidney#imid_21034227

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA006308; green = DDAH1, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for DDAH1 IHC & IF

🧪Run adrenal gland first: glandular cells should stain (HPA: High in adrenal gland glandular cells). Use adipose tissue as the negative tissue (HPA: Not detected in adipocytes); on the adrenal slide, assess non-glandular cells as an internal background reference, without assuming they are DDAH1-negative (HPA: adrenal gland entry specifies glandular cells only).

Positive control tissue: Adrenal gland (Glandular cells, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show DDAH1 in A-431, U2OS, with annotated localisation: Plasma membrane (approved), Cytosol (approved) (HPA subcellular).

Technical controls: Include no-primary (secondary-only), a rabbit IgG isotype control matched to the primary antibody’s clonality, and DDAH1 knockout tissue if available (caption: rabbit primary; standard IHC controls). For chromogenic staining, block endogenous peroxidase and check endogenous biotin when using the caption’s biotin-based detection; distinguish adrenal pigment from DAB signal (caption: biotin-based detection with DAB; standard IHC practice).

⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected PB10000 tissue-IHC caption does not state the fixative (caption: fixative not stated). The caption uses heat-mediated citrate retrieval at pH 6 for 20 minutes, but does not establish whether retrieval is required (caption: rat brain paraffin-section IHC). The supplied evidence does not establish that frozen sections or IF are easier; interpret brown signal in adrenal tissue with attention to native pigment (standard IHC practice).

HPA tissue IHC evidence for DDAH1

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.

Positive expression · recommended positive controls

Tissue	Cell type	Level	Evidence	Source
Adrenal gland	Glandular cells	High	Protein (IHC)	HPA →
Bronchus	Respiratory epithelial cells	High	Protein (IHC)	HPA →
Cerebral cortex	Glial cells	High	Protein (IHC)	HPA →
Fallopian tube	Glandular cells	High	Protein (IHC)	HPA →
Gallbladder	Glandular cells	High	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 →
Cerebellum	Cells in granular layer	Not detected	Protein (IHC)	HPA →
Cervix	Squamous epithelial cells	Not detected	Protein (IHC)	HPA →
Endometrium	Cells in endometrial stroma	Not detected	Protein (IHC)	HPA →

Section 3

Advanced DDAH1 IHC Tips

Troubleshoot DDAH1 staining in paraffin sections by checking retrieval, cell type and compartment before comparing chromogenic signal across samples.

How should I adjust retrieval when DDAH1 staining is weak or uneven?

Start with heat-mediated retrieval in citrate buffer at pH 6 for 20 minutes (datasheet PB10000). That condition preceded DDAH1 staining in a paraffin-embedded rat brain section, with 10% goat serum block and 1 μg/mL primary antibody overnight at 4°C (datasheet PB10000). If signal is weak, check heating consistency and section adhesion before testing a different retrieval condition on matched sections (standard IHC practice). Compare changes against the same positive tissue and a no-primary control, because stronger DAB staining alone cannot establish improved DDAH1 specificity (standard IHC practice).

Could fixation explain weak DDAH1 staining in my paraffin sections?

DDAH1-specific sensitivity to fixation is unknown: the selected paraffin-section caption does not state a fixative (datasheet PB10000). Record each sample’s fixative and fixation duration, then compare sections processed together using citrate retrieval at pH 6 for 20 minutes (datasheet PB10000; standard IHC practice). In a controlled comparison, keep section thickness, primary-antibody exposure and DAB development consistent so a staining difference can be assessed against the fixation variable (standard IHC practice). Do not infer a DDAH1 fixation effect from tissue expression patterns or from its annotated modifications, including S-nitrosocysteines at residues 222 and 274 (HPA: tissue IHC; UniProt O94760).

Where should DDAH1 staining appear, and when is nuclear signal suspicious?

Assess cytoplasmic staining first: tissue IHC reports cytoplasmic expression in most tissues, including kidney (HPA: tissue IHC). In kidney, proximal-tubule cell bodies show high staining, making cell identity useful when assessing a weak or patchy DAB pattern (HPA: High in kidney proximal tubules). Cell imaging additionally places DDAH1 mainly in cytosol and at the plasma membrane, with nucleoli as an additional location; these are complementary localisation observations, not a tissue-IHC scoring rule (HPA: subcellular). If staining appears only in nuclei or along section edges, compare morphology and no-primary controls before calling it DDAH1 (standard IHC practice).

Can this antibody distinguish DDAH1 isoforms or a modified epitope?

DDAH1 has 2 listed isoforms, but the supplied antibody evidence does not map its epitope or establish isoform selectivity (UniProt O94760; datasheet PB10000). The protein has no annotated transmembrane segment, and its listed modifications include N-acetylalanine at residue 2 and S-nitrosocysteines at residues 222 and 274 (UniProt O94760). Therefore, neither cytoplasmic DAB staining nor a change after citrate retrieval at pH 6 identifies which isoform or modification was detected (UniProt O94760; datasheet PB10000; standard IHC interpretation). Resolve that question with documented epitope mapping or independently validated isoform-specific reagents before assigning the staining to one molecular form (standard IHC practice).

How can IF help assess the cell type and compartment behind IHC staining?

Use IF/ICC as a complementary localisation check, pairing DDAH1 with a validated marker for the expected cell type, such as a proximal-tubule marker when investigating kidney staining (HPA: High in kidney proximal tubules; standard IF practice). Choose spectrally separated fluorophores and inspect unstained tissue in each channel so autofluorescence is not mistaken for DDAH1 signal (standard IF practice). Permeabilise when testing cytosolic or nucleolar signal, but determine access requirements for a proposed membrane epitope from its mapping rather than assuming an extracellular side (HPA: subcellular; UniProt O94760 topology; standard IF practice). Compare the IF pattern with chromogenic IHC cautiously, since the selected antibody caption documents paraffin-section IHC, not an IF protocol (datasheet PB10000).

What should I check when DAB obscures weak DDAH1 staining?

Run a no-primary control and inspect whether DAB deposits follow tissue architecture or occur diffusely across the section (standard IHC practice). The selected workflow used 10% goat serum, biotinylated goat anti-rabbit secondary antibody for 30 minutes at 37°C, a streptavidin-biotin complex and DAB (datasheet PB10000). Include a peroxidase block and assess endogenous biotin when using that detection system; optimise blocking and washing on matched sections if background persists (standard IHC practice). Preserve the same DAB development time across comparisons, since longer development can make nonspecific deposits look like an increase in DDAH1 staining (standard IHC practice).

How should I quantify DDAH1 IHC across samples with different cell mixtures? ⚠ ANSWER MARKED FOR VERIFICATION

Define the scored compartment and cell population before measuring DAB, using cytoplasmic signal as the tissue-IHC reference pattern (HPA: tissue IHC). In kidney, score proximal-tubule cell bodies separately from other structures because that population has high reported staining (HPA: High in kidney proximal tubules). Report either an H-score from staining intensity and percentage of positive cells, or positive-cell density per mm²; normalise to the number or area of eligible cells, not total section area when cell composition differs (standard IHC practice). Keep retrieval at pH 6 for 20 minutes and detection settings consistent across the scored sections (datasheet PB10000; standard IHC practice).

How can I distinguish convincing DDAH1 signal from an IHC artefact?

A convincing result aligns with plausible cells and compartments: kidney proximal-tubule cell bodies stain strongly, and tissue IHC broadly describes cytoplasmic DDAH1 (HPA: tissue IHC). Treat signal confined to cut edges, damaged or necrotic areas, or unexpected cells as provisional until morphology and controls support it (standard IHC practice). Check no-primary staining and peroxidase-related DAB deposition before attributing brown deposits to DDAH1, especially with chromogenic detection (standard IHC practice). Cell imaging also reports plasma-membrane and additional nucleolar localisation, so an unusual compartment merits independent confirmation rather than automatic rejection; tissue-IHC and cell-imaging evidence answer different localisation questions (HPA: subcellular; HPA: tissue IHC).

Boster reagents

Best DDAH1 / N(G),N(G)-dimethylarginine dimethylaminohydrolase 1 IHC Antibodies

PB10000 has illustrated paraffin-section IHC in human, mouse and rat brain and IF in U20S cells (PB10000 image captions). M03136 lists IHC and IF in those species plus monkey (M03136 catalog applications/reactivity).

Real IHC data

Anti-DDAH1 Antibody ®

Cat # PB10000

View product →

PB10000 lists IHC and IF, with illustrated paraffin-section IHC in human, mouse and rat brain and IF in U20S cells (PB10000 catalog applications/image captions). M03136 lists IHC and IF with human, monkey, mouse and rat reactivity; no IHC or IF image is supplied for it (M03136 catalog applications/reactivity/image alts).

Which to pick: For tissue IHC, choose PB10000 when an illustrated paraffin-section example matters; its human, mouse and rat brain captions do not report the fixative (PB10000 IHC image captions). For IF/ICC, PB10000 has an illustrated U20S-cell example, while M03136 lists IF/ICC without an image in this payload (PB10000 IF image caption; M03136 catalog applications/image alts). For the broadest listed species coverage, choose rabbit monoclonal M03136, clone AEAG-4, which includes monkey alongside human, mouse and rat (M03136 catalog host/clone/reactivity).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry O94760 (DDAH1_HUMAN, N(G),N(G)-dimethylarginine dimethylaminohydrolase 1).
Human Protein Atlas. DDAH1 tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. DDAH1 subcellular location (ICC-IF): Mainly localized to the plasma membrane and cytosol. In addition localized to the nucleoli..
Human Protein Atlas. DDAH1 antibody validation summary (2 antibodies).
Dimethylarginine dimethylaminohydrolase-2 deficiency promotes vascular regeneration and attenuates pathological angiogenesis. Experimental eye research 2016 — PMC4912010.
DDAH1 Promotes Cisplatin Chemoresistance in Patients with Locally Advanced Nasopharyngeal Carcinoma via the EGFR-JAK2-STAT3 Pathway. Advanced science (Weinheim, Baden-Wurttemberg, Germany) 2025 — PMC12376576.
Involvement of DDAH/ADMA/NOS/cGMP and COX-2/PTGIS/cAMP Pathways in Human Tissue Kallikrein 1 Protecting Erectile Function in Aged Rats. PloS one 2017 — PMC5245815.
DDAH1 mediates gastric cancer cell invasion and metastasis via Wnt/β-catenin signaling pathway. Molecular oncology 2017 — PMC5579338.
PubMed PMID:9874257 — UniProt-cited evidence.
PubMed PMID:10493931 — UniProt-cited evidence.
PubMed PMID:17974005 — UniProt-cited evidence.