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DNPH1 / 5-hydroxymethyl-dUMP N-hydrolase · IHC design guide
Design Immunohistochemistry for DNPH1
Plan DNPH1 chromogenic IHC in paraffin sections around the predominantly cytoplasmic tissue pattern (HPA tissue IHC). Use high-staining duodenal glandular cells and kidney proximal tubules as positive references, and score staining by cell type and compartment (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
DNPH1 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 tissue staining; nuclear location annotated (HPA tissue IHC; UniProt) | |
| Staining pattern | Mostly cytoplasmic; high in duodenal glandular cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A30621) | |
| Positive control | Duodenum+4 more · see all | |
| Negative control | Adipose tissue+2 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Splenic red pulp is unstained despite reported high splenic expression (HPA tissue IHC; UniProt) | |
| Regulation | Overexpressed in some breast cancers (UniProt) | |
| Isoform / epitope | 2 isoforms; check antibody epitope coverage (UniProt) |
Section 1
Recommended DNPH1 IHC & IF Protocols
The catalog antibody has an IHC-P protocol (datasheet A30621). One published DNPH1 IHC protocol for human tissue microarrays is shown below (PMC12990376).
| Sample | Paraffin-embedded human uterus tissue; fixative not specified (datasheet A30621) |
| Fixation | Image fixative and duration unreported (datasheet A30621); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Tris-EDTA pH 9.0 (datasheet A30621); 20 min, 95–100 °C (standard) |
| 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-DNPH1, 1:50 recommended; image 1:200 (datasheet A30621) |
| 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 | DNPH1-positive staining in glandular cells of duodenum (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 heat retrieval for the catalog antibody (datasheet A30621); the published protocol used a 20 min alternative (PMC12990376).
Section 2
What Is the Expected DNPH1 Staining Pattern?
In paraffin-section IHC, expect predominantly cytoplasmic DNPH1 staining in glandular cells of duodenum, gallbladder and small intestine, and in kidney proximal tubules (HPA tissue IHC: High; cytoplasmic expression in most tissues). UniProt also places DNPH1 in the nucleus and reports no transmembrane segment (UniProt O43598). HPA rates tissue IHC reliability Enhanced, while noting medium consistency between antibody staining and RNA expression (HPA tissue IHC).
| Clear cytoplasmic staining in kidney proximal-tubule cell bodies or glandular cells of duodenum, gallbladder or small intestine. | This fits the reported high-staining cell populations and the predominantly cytoplasmic tissue pattern (HPA tissue IHC: High; cytoplasmic expression in most tissues). Interpret intensity within the named cells, rather than assigning one score to the whole section (general IHC practice). |
| Predominantly nuclear staining in paraffin-section IHC, with little staining in the expected cytoplasmic compartment. | This differs from the reported tissue IHC pattern (HPA tissue IHC: cytoplasmic expression in most tissues). Nuclear DNPH1 remains plausible because UniProt lists the nucleus and HPA ICC-IF reports mainly nucleoplasmic localization (UniProt O43598; HPA subcellular). Check the IHC controls before calling it an artifact (general IHC practice). |
| Strong staining in adipocytes, chondrocytes or splenic red-pulp cells, especially when expected positive cells are weak. | Those cell populations were reported as not detected (HPA tissue IHC). Consider cross-reactivity or endogenous detection activity, then inspect the no-primary control and expected positive tissue (general IHC practice). A different specimen is not automatically a definitive DNPH1-negative control. |
| Diffuse chromogen across cells, extracellular space or the whole section, obscuring cell boundaries. | A widespread haze is difficult to reconcile with the reported cell-resolved cytoplasmic pattern (HPA tissue IHC). Review blocking, washes, detection reagents and chromogen development with controls (general IHC practice); the appearance alone cannot identify which step caused the background. |
| No convincing stain in kidney proximal tubules or high-staining glandular cells. | Absence in these reported positive populations makes the run inconclusive before it supports a biological negative call (HPA tissue IHC: High). Check tissue preservation, antibody dilution, retrieval and detection performance against the run controls (general IHC practice); no DNPH1-specific fixation sensitivity is reported here. |
💡Expected DNPH1 appearanceCall a positive IHC result when cytoplasmic staining is discernible in high-staining glandular cells or kidney proximal-tubule cell bodies (HPA tissue IHC: High); diffuse haze or isolated strong staining in reported negative cells calls for control review (HPA tissue IHC: Not detected; general IHC practice).
| Assay-specific localization | Q: Should the IHC pattern define the IF/ICC result? A: No. Tissue IHC is predominantly cytoplasmic, whereas ICC-IF is mainly nucleoplasmic with additional cytosol signal (HPA tissue IHC; HPA subcellular). UniProt lists both cytoplasm and nucleus (UniProt O43598). Interpret the assays in their own specimens. |
| Strength of the IHC evidence | Two listed rabbit polyclonal antibodies have Enhanced IHC status (HPA antibodies: HPA029675, HPA029676). The tissue profile is also rated Enhanced, with medium consistency against RNA expression (HPA tissue IHC). Use that support for the reported pattern, while evaluating each new staining run with its controls (general IHC practice). |
| Cell-level tissue variation | High staining is reported for selected glandular cells and kidney proximal tubules; adrenal glandular and bronchial respiratory epithelial cells are Medium (HPA tissue IHC). Adipocytes and chondrocytes are Not detected (HPA tissue IHC). Compare the relevant cell population rather than treating every cell in an organ as equivalent. |
| Protein abundance versus tissue staining | UniProt describes high expression in spleen, yet HPA reports no detectable staining in splenic red-pulp cells (UniProt O43598 tissue specificity; HPA tissue IHC: Not detected). These statements concern different measurements and cell scopes; do not designate red pulp as an IHC positive solely from the UniProt tissue-level description. |
| Isoforms and antibody coverage | Two DNPH1 isoforms are listed, but the supplied records give no antibody epitope coordinates or isoform-specific staining results (UniProt O43598: isoforms 1 and 2; HPA antibodies). Do not infer that a stained or unstained cell identifies one isoform, or that both catalog antibodies detect each isoform equally. |
| Topology and processing | DNPH1 has no annotated transmembrane segment or signal peptide; its annotated chain is residues 2–174 (UniProt O43598 topology and processing). A sharply membrane-restricted pattern would therefore need scrutiny, but these annotations do not establish an epitope location or any DNPH1-specific effect of fixation or antigen retrieval. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive cells are blank in the test section. | The run may have lost detectable signal; the cause cannot be assigned from one negative slide (general IHC practice). | Check a section containing kidney proximal tubules or reported high-staining glandular cells (HPA tissue IHC: High). Review antibody dilution, retrieval, detection and section quality against run controls (general IHC practice). |
| Most cells show broad brown haze or deposit. | Excess background can obscure the reported cytoplasmic pattern (HPA tissue IHC; general IHC practice). | Compare with a no-primary control, then review blocking, washing and chromogen development one step at a time (general IHC practice). Reassess whether cell borders and the expected compartment can be resolved. |
| A reported negative cell population stains strongly. | Possible nonspecific binding or endogenous detection activity (general IHC practice); adipocytes, chondrocytes and splenic red-pulp cells were Not detected (HPA tissue IHC). | Examine the no-primary control and a reported positive population in the same run; verify that the signal tracks the expected cell type and compartment before scoring it (HPA tissue IHC; general IHC practice). |
| Nuclear signal dominates the IHC section. | The pattern differs from predominantly cytoplasmic tissue IHC, although nuclear localization is documented by UniProt and ICC-IF (HPA tissue IHC; UniProt O43598; HPA subcellular). | Compare positive and no-primary controls, inspect morphology and report nuclear staining separately from cytoplasmic staining (general IHC practice). Do not treat the ICC-IF pattern as an IHC acceptance criterion. |
| The spleen seems inconsistent with the expected positive tissue list. | UniProt reports high spleen expression, while HPA found no staining in splenic red-pulp cells (UniProt O43598 tissue specificity; HPA tissue IHC: Not detected). | Identify the splenic cell population being scored and use an HPA high-staining IHC population as the positive comparator (HPA tissue IHC). Avoid interpreting a red-pulp negative as assay failure by itself. |
| Two antibody runs give different compartment or cell-type patterns. | Both listed antibodies have Enhanced IHC status, but that status does not establish identical staining in a new run (HPA antibodies; general IHC practice). | Compare matched tissue areas and controls, then assess each result against the reported cytoplasmic cell pattern (HPA tissue IHC; general IHC practice). Record the disagreement rather than assigning it to an unreported isoform or fixation effect. |
Experiment evidence
IHC & IF images showing DNPH1 staining
A30621 · IHCImmunohistochemical analysis of paraffin-embedded human uterus. 1, Antibody was diluted at 1:200(4° overnight). 2, Tris-EDTA,pH9.0 was used
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000112667-DNPH1/tissue/cerebral+cortex#imid_7413616
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000112667-DNPH1/tissue/colon#imid_7411878
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000112667-DNPH1/tissue/liver#imid_7411879
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000112667-DNPH1/tissue/kidney#imid_7411880
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA029675; green = DNPH1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA029675; green = DNPH1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for DNPH1 IHC & IF
🧪Run kidney first; proximal tubule cell bodies should stain (HPA: High in kidney proximal tubules). Run adipose tissue as the negative tissue control (HPA: Not detected in adipocytes); on the kidney slide, cells outside proximal tubules should show only background staining if used as internal negatives, but their DNPH1 status needs independent validation (HPA: kidney row specifies proximal tubules only).
Positive control tissue: Duodenum (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show DNPH1 in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host and class, or host-matched nonimmune IgG for a polyclonal antibody (standard IHC practice). Use a verified DNPH1 knockout specimen or a documented immunizing-peptide block as a biological specificity control, and check endogenous peroxidase and biotin signal in kidney, particularly proximal tubules (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence; the selected paraffin-section caption also leaves the fixative unreported (A30621 tissue-IHC caption: fixative not stated). The caption uses Tris-EDTA at pH 9.0 for antigen retrieval, but does not establish that retrieval is required (A30621 tissue-IHC caption). Paraffin IHC has a supplied example, while the evidence does not establish whether frozen sections or IF are easier; kidney proximal tubules warrant a check for endogenous biotin signal (A30621 tissue-IHC caption; HPA: kidney proximal tubules High; standard IHC practice).
HPA tissue IHC evidence for DNPH1
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 |
|---|---|---|---|---|
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Kidney | Proximal tubules (cell body) | High | Protein (IHC) | HPA → |
| Small intestine | Glandular cells | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced DNPH1 IHC Tips
Troubleshoot DNPH1 staining in paraffin sections by checking retrieval, cellular distribution, controls and scoring before interpreting differences between tissues.
How should I adjust retrieval if DNPH1 staining is weak or uneven?
Use heat-mediated Tris-EDTA at pH 9.0 for DNPH1 antigen retrieval (datasheet A30621). The selected paraffin-section example used antibody at 1:200 overnight at 4°C, so compare retrieval changes while holding that primary incubation constant (caption A30621). Check whether weak staining also affects the section interior, because uneven heating or drying can create misleading gradients (standard IHC practice). Include a high-staining reference area such as kidney proximal tubules to judge whether retrieval worked (HPA: High in kidney proximal tubules). If staining remains weak, trial a gentler or stronger heat exposure on matched sections and record any tissue damage (standard IHC practice).
Could fixation explain weak DNPH1 staining in my paraffin sections?
DNPH1-specific sensitivity to fixation is unknown from the supplied evidence; the selected paraffin-section caption does not state a fixative (caption A30621). Record the fixative, fixation duration and tissue thickness for each specimen, then compare sections processed together (standard IHC practice). Overfixation can reduce epitope accessibility in IHC generally, but no DNPH1-specific fixation effect is established here (standard IHC practice). Keep retrieval at Tris-EDTA pH 9.0 while testing a fixation variable, so changes in staining are interpretable (datasheet A30621; standard IHC practice). Use a same-run positive control to distinguish specimen processing from a failed staining run (standard IHC practice).
Should DNPH1 stain nuclei, cytoplasm, or both in tissue IHC?
Expect predominantly cytoplasmic staining across many tissues in chromogenic IHC, while allowing a nuclear component because DNPH1 is annotated in both cytoplasm and nucleus (HPA: cytoplasmic expression in most tissues; UniProt O43598: cytoplasm and nucleus). HPA cell imaging places DNPH1 mainly in nucleoplasm with additional cytosol, so the relative signal can depend on assay and cell context (HPA subcellular: nucleoplasm approved; cytosol supported). DNPH1 has no transmembrane segment; an exclusively crisp membrane rim warrants scrutiny (UniProt O43598: topology). Assess nuclei against the counterstain and inspect cytoplasm within the same cells (standard IHC practice). Compare the pattern with kidney proximal tubules as a tissue reference (HPA: High in kidney proximal tubules).
Could DNPH1 isoforms or modifications change the IHC pattern?
DNPH1 has 2 annotated isoforms, so an antibody's epitope must be checked against each sequence before treating a staining difference as isoform-specific (UniProt O43598: isoforms 1 and 2; standard antibody validation practice). The supplied caption gives an IHC dilution and retrieval condition but no epitope map (caption A30621). Annotated modifications include N-acetylalanine at residue 2 and phosphoserines at 28, 98, 123 and 128; their effects on this antibody are unknown (UniProt O43598: modified residues). Compare epitope documentation with isoform sequences and modification sites if available (standard antibody validation practice). Without that mapping or an orthogonal control, report the staining as DNPH1 immunoreactivity rather than assigning an isoform or modification state (standard IHC practice).
How can I compare DNPH1 IHC with multiplex IF?
Treat IF as a separate assay: the selected product evidence describes paraffin-section IHC with Tris-EDTA pH 9.0 retrieval, not an IF protocol (caption A30621). Multiplex DNPH1 with a validated marker of the cell population being assessed, such as a proximal-tubule marker when examining kidney (HPA: High in kidney proximal tubules; standard IF practice). Choose a fluorophore channel with low tissue autofluorescence and include unstained and single-stain controls to assess bleed-through (standard IF practice). Because DNPH1 is intracellular and has no transmembrane segment, permeabilise cells or sections as needed to expose cytoplasmic and nuclear epitopes, then optimise against morphology (UniProt O43598: localisation and topology; standard IF practice). Compare compartment patterns cautiously: HPA IF reports mainly nucleoplasm with additional cytosol (HPA subcellular).
What should I check when DNPH1 chromogenic staining is diffuse?
First compare the diffuse signal with a no-primary control and the counterstained tissue architecture (standard IHC practice). Block endogenous peroxidase for a peroxidase-based chromogenic assay, then assess whether residual colour persists without primary antibody (standard IHC practice). Optimise protein blocking, washing and primary-antibody concentration on matched sections; 1:200 is the dilution in the selected paraffin-section example, not a universal optimum (caption A30621; standard IHC practice). DNPH1 is reported as cytoplasmic in most tissues, so broad cytoplasmic staining alone does not establish nonspecific binding (HPA tissue IHC profile). Examine adipocytes or chondrocytes as low-signal comparators where present, while recognising that morphology and processing can affect their appearance (HPA: Not detected in adipocytes and chondrocytes; standard IHC practice).
How should I score DNPH1 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because HPA reports cytoplasmic tissue staining while cell imaging also supports nucleoplasmic localisation (HPA tissue IHC profile; HPA subcellular). For a defined population, record the percentage of positive cells and staining intensity; an H-score can combine intensity categories with their percentages (standard IHC scoring practice). For spatial comparisons, report positive-cell density per mm² of viable tissue or the fraction of positive cells among all eligible cells (standard IHC scoring practice). Normalise to the same eligible cell type, compartment, section area and staining batch across samples (standard IHC practice). Exclude folds, necrosis and edge artefacts using prespecified rules, and retain representative images alongside the scores (standard IHC practice).
How do I distinguish genuine DNPH1 staining from an artefact?
A credible pattern should follow cell boundaries and plausible DNPH1 compartments: cytoplasm in tissue IHC, with possible nuclear staining (HPA tissue IHC profile; UniProt O43598: localisation). Compare candidate positive cells with reported high-staining kidney proximal tubules or intestinal glandular cells, and use adipocytes as a reported undetected comparator where available (HPA: High in kidney proximal tubules and small-intestine glandular cells; Not detected in adipocytes). An isolated membrane rim is difficult to reconcile with the absence of a transmembrane segment (UniProt O43598: topology). Discount colour concentrated at section edges, folds or necrotic areas, and investigate signal surviving the no-primary control as possible endogenous enzyme activity (standard IHC practice). HPA's tissue staining has medium RNA concordance, so confirm consequential claims with an independent control (HPA: Enhanced reliability, medium consistency).
Boster reagents
Best DNPH1 / 5-hydroxymethyl-dUMP N-hydrolase IHC Antibodies
Three DNPH1/RCL catalog antibodies list human IHC (catalog: applications/reactivity). Only A30621 has an IHC figure of paraffin-embedded human uterus (A30621 image caption); none lists IF/ICC (catalog: applications).
Real IHC data
Anti-RCL Antibody
Cat # A30621
A30621 lists IHC for human, mouse and rat and has an IHC image of paraffin-embedded human uterus (A30621 catalog: applications/reactivity; image caption). M08676 and M08676-1 list human IHC, but neither has an IHC image caption (catalog: applications/reactivity; image fields).
Which to pick: For tissue IHC, choose A30621 when an image from paraffin-embedded human uterus is relevant; its caption reports 1:200 primary antibody overnight at 4 °C and Tris-EDTA retrieval at pH 9.0, but does not report the fixative (A30621 image caption). For cross-species work, A30621 lists human, mouse and rat reactivity, while the monoclonals M08676 (clone 27D38) and M08676-1 list human reactivity only (catalog: reactivity/clone). No listed SKU has IF/ICC validation, so there is no catalog-supported IF/ICC pick (catalog: applications; IF image fields).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O43598 (DNPH1_HUMAN, 5-hydroxymethyl-dUMP N-hydrolase).
- Human Protein Atlas. DNPH1 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. DNPH1 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the cytosol..
- Human Protein Atlas. DNPH1 antibody validation summary (2 antibodies).
- Promotion of breast cancer by the DNPH1 enzyme. iScience 2026 — PMC12990376.
- PubMed PMID:9271375 — UniProt-cited evidence.
- PubMed PMID:18726892 — UniProt-cited evidence.
- PubMed PMID:14574404 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for DNPH1 (5-hydroxymethyl-dUMP N-hydrolase). Expected localisation Cytoplasmic tissue staining; nuclear location annotated (HPA tissue IHC; UniProt). Antigen retrieval: Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A30621). Validated IHC antibody: A30621 at 1:50 recommended; image 1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Duodenum (Glandular cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human uterus tissue; fixative not specified (datasheet A30621); 2. Fixation: Image fixative and duration unreported (datasheet A30621); 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: Tris-EDTA pH 9.0 (datasheet A30621); 20 min, 95–100 °C (standard); 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-DNPH1, 1:50 recommended; image 1:200 (datasheet A30621); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Splenic red pulp is unstained despite reported high splenic expression (HPA tissue IHC; UniProt); Isoform / epitope: 2 isoforms; check antibody epitope coverage (UniProt); Regulation: Overexpressed in some breast cancers (UniProt). Evidence: UniProt O43598, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A30621, PMC12990376. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/DNPH1/immunohistochemistry-ihc-guide.