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OTUD5 / OTU domain-containing protein 5 · IHC design guide
Design Immunohistochemistry for OTUD5
Plan chromogenic OTUD5 IHC-P using 2.5 μg/mL as a starting antibody concentration (datasheet: 2.5 μg/mL). Compare cytoplasmic tissue staining (HPA tissue IHC) with the annotated nuclear location (UniProt), and use controls to assess specificity.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
OTUD5 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 (HPA tissue IHC); nuclear location annotated (UniProt) | |
| Staining pattern | Cytoplasmic staining in glandular cells and colon endothelium (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Epididymis+4 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 A09744) | |
| Caveat | Observed cytoplasmic staining differs from the nuclear annotation (HPA tissue IHC; UniProt) | |
| Regulation | Expression regulation is not established (UniProt) | |
| Isoform / epitope | Five isoforms; epitope coverage is unresolved (UniProt) |
Section 1
Recommended OTUD5 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published OTUD5 IHC methods from bladder cancer, liver, and tissue-section studies (PMC9463452; PMC10613150; PMC13559827).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A09744); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-OTUD5, 2.5 μg/mL (datasheet A09744) |
| 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 | OTUD5-positive staining in glandular cells of appendix (HPA tissue IHC: Medium). HPA tissue profile: General cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA HIER at pH 9.0, 95–98 °C for 20 min (page retrieval); citrate pH 6.0 is a published alternative (PMC13559827).
Section 2
What Is the Expected OTUD5 Staining Pattern?
For paraffin-section IHC, expect mainly cytoplasmic OTUD5 staining, including medium staining in glandular cells of appendix, breast, cervix, duodenum, endometrium, and gallbladder (HPA tissue IHC). OTUD5 has no transmembrane segment (UniProt Q96G74 topology). UniProt lists the nucleus, while HPA reports general cytoplasmic IHC expression; HPA rates its tissue IHC evidence Approved, with medium staining–RNA consistency and external verification pending (UniProt Q96G74; HPA tissue IHC).
| Cytoplasmic chromogen in glandular cells of appendix or duodenum, with discernible cell boundaries and limited background (HPA tissue IHC). | This fits the reported compartment and a medium-staining cell population (HPA tissue IHC). Compare cells within the same section before scoring: scattered pale deposits outside cell boundaries should not be counted as OTUD5-positive cells (general IHC practice). |
| A crisp membrane rim is the dominant signal, with little cytoplasmic staining. | A membrane-only pattern conflicts with general cytoplasmic tissue staining and the absence of a transmembrane segment (HPA tissue IHC; UniProt Q96G74 topology). Treat it as a possible artefact and review antibody concentration, detection controls, and tissue morphology before interpreting it as OTUD5 (general IHC practice). |
| Strong staining appears in cardiomyocytes or smooth muscle cells, which HPA lists as not detected (HPA tissue IHC). | An unexpected cell-type pattern raises concern for cross-reactivity or endogenous detection activity; it does not by itself identify the cause (general IHC practice). Compare a known-positive cell population and a primary-antibody omission control under the same detection conditions (HPA tissue IHC; general IHC practice). |
| Brown precipitate covers stroma, empty spaces, and cell surfaces without a clear cellular pattern. | Diffuse deposition is background rather than a scoreable cytoplasmic pattern (general IHC practice; HPA tissue IHC). Check the primary-antibody omission control, washing, blocking, and chromogen development; assign a cell-level score only where staining can be localized (general IHC practice). |
| Glandular cells in appendix or duodenum show no signal, despite medium staining reported there (HPA tissue IHC). | First check whether another expected-positive section stained in the same run (HPA tissue IHC; general IHC practice). Review retrieval conditions, antibody dilution, detection reagents, and tissue preservation as general workflow variables; this record does not establish OTUD5-specific fixation sensitivity (general IHC practice). |
💡Expected OTUD5 appearanceCall positive when glandular cells show interpretable, mainly cytoplasmic staining of roughly medium intensity in an HPA-listed medium tissue; membrane-only rims or diffuse extracellular deposits are suspect patterns (HPA tissue IHC; UniProt Q96G74 topology; general IHC practice).
| Compartment evidence | HPA describes general cytoplasmic tissue IHC staining, whereas UniProt lists nuclear localization (HPA tissue IHC; UniProt Q96G74). Record nuclear signal separately instead of assuming that either source proves every nuclear or cytoplasmic deposit specific (general IHC practice). |
| Tissue and cell selection | Appendix and duodenal glandular cells are reported at medium intensity; cardiomyocytes and smooth muscle cells are listed as not detected (HPA tissue IHC). These provide practical comparison populations, although a not-detected HPA entry is not proof that every specimen will be blank (HPA tissue IHC; general IHC practice). |
| Strength of IHC evidence | The HPA tissue profile is Approved, but staining and RNA have medium consistency and external verification is pending (HPA tissue IHC). The listed HPA017375 antibody is IHC Approved, not IHC Enhanced (HPA antibodies). Interpret an unexpected pattern with controls rather than treating approval as independent confirmation in every tissue (general IHC practice). |
| IF/ICC Q: Where should OTUD5 appear? | A: HPA reports mainly cytosolic staining, with additional nucleoplasm and nucleoli fibrillar-center localization in ICC-IF; its main and additional locations are Approved (HPA subcellular ICC-IF). This describes IF/ICC images and does not replace the cytoplasmic tissue-IHC expectation (HPA tissue IHC). |
| Isoform coverage | UniProt lists five OTUD5 isoforms and an OTU domain at residues 213–341 (UniProt Q96G74). The supplied evidence gives no antibody epitope or isoform-recognition map, so a staining difference cannot be assigned to a particular isoform from this record (UniProt Q96G74; HPA antibodies). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected-positive glandular cells remain blank (HPA tissue IHC). | A failed IHC run or weak detection is possible; the supplied sources do not identify an OTUD5-specific retrieval requirement (general IHC practice). | Check a same-run positive section, reagent integrity, antibody dilution, and retrieval conditions using the applicable antibody instructions; document any change before comparing intensity (general IHC practice). |
| Every compartment has faint, even chromogen. | Nonspecific primary or detection background may obscure the reported cytoplasmic pattern (HPA tissue IHC; general IHC practice). | Compare primary-antibody omission and detection controls; review blocking, washes, and development time, then score only localized cellular staining (general IHC practice). |
| Nuclear staining dominates while cytoplasm is blank. | UniProt lists nuclear localization, and ICC-IF includes nuclear sites, but HPA tissue IHC describes general cytoplasmic expression (UniProt Q96G74; HPA subcellular ICC-IF; HPA tissue IHC). | Record the compartment and assess a positive tissue and detection controls; do not score nuclear-only IHC as the expected cytoplasmic pattern without further validation (HPA tissue IHC; general IHC practice). |
| Unexpected strong staining appears in an HPA not-detected cell population (HPA tissue IHC). | Cross-reactivity or endogenous detection activity is possible, particularly when the signal appears independently of the primary antibody (general IHC practice). | Run a primary-antibody omission control and, for peroxidase detection, check endogenous peroxidase blocking; compare morphology and the expected-positive cells (general IHC practice; HPA tissue IHC). |
| Signal forms a sharp membrane outline. | The pattern conflicts with HPA's general cytoplasmic IHC profile and UniProt's lack of a transmembrane segment (HPA tissue IHC; UniProt Q96G74 topology). | Inspect the omission control, tissue edges, chromogen development, and primary-antibody dilution before assigning OTUD5 positivity (general IHC practice). |
| Adjacent sections give different apparent intensities. | Cell composition and staining-run variation can change the comparison; HPA lists medium staining in selected cell populations rather than a uniform tissue-wide signal (HPA tissue IHC; general IHC practice). | Compare the same cell type and compartment across sections, retain a same-run control, and report intensity separately from the fraction of positive cells (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing OTUD5 staining
A09744 · IHCImmunohistochemistry of OTUD5 in mouse kidney tissue with OTUD5 antibody at 2.5 μg/mL.
A09744 · IFImmunofluorescence of OTUD5 in Human Kidney cells with OTUD5 antibody at 20 μg/mL.
A09744-1 · IFIF analysis of OTUD5 using anti-OTUD5 antibody (A09744-1) and anti-Beta Tubulin antibody (M01857-3). OTUD5 was detected in immunocytochemica
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000068308-OTUD5/tissue/cerebral+cortex#imid_4683676
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000068308-OTUD5/tissue/colon#imid_4683594
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000068308-OTUD5/tissue/liver#imid_4683603
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000068308-OTUD5/tissue/kidney#imid_4683596
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA017375; green = OTUD5, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA017375; green = OTUD5, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for OTUD5 IHC & IF
🧪Run appendix first and expect glandular cells to stain (HPA: Medium in appendix glandular cells). Use epididymis glandular cells as a tissue negative (HPA: Not detected); on the appendix slide, treat adjacent cells that remain unstained as internal background, since HPA does not identify a validated negative cell type there (HPA: appendix glandular-cell row).
Positive control tissue: Appendix (Glandular cells, HPA Medium)
Negative control tissue: Epididymis (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show OTUD5 in A-431, U-251MG, U2OS, with annotated localisation: Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide and an isotype control matched to the primary antibody’s host species and clonality; use OTUD5 knockout tissue or cells as a biological negative (standard IHC practice). Quench endogenous peroxidase and check for background chromogen in the appendix section (standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected A09744 mouse-kidney IHC caption does not state a fixative (A09744 tissue-IHC caption). Antigen-retrieval dependence is unreported, so optimize retrieval empirically for paraffin sections (supplied target/application evidence; standard IHC practice). HPA shows ICC-IF images in A-431, U-251MG and U2OS, but the supplied evidence does not establish that IF or frozen sections are easier; distinguish glandular-cell signal from luminal background when scoring appendix (HPA: subcellular ICC-IF cell lines; HPA: Medium in appendix glandular cells; standard IHC practice).
HPA tissue IHC evidence for OTUD5
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium 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 |
|---|---|---|---|---|
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Colon | Endothelial cells | Medium | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Epididymis | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Fallopian tube | Ciliated cells (cell body) | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Seminal vesicle | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced OTUD5 IHC Tips
Troubleshoot OTUD5 staining in paraffin sections by checking retrieval, cellular distribution, controls and scoring before interpreting chromogenic signal.
How should I troubleshoot weak OTUD5 staining after antigen retrieval?
Use Tris-EDTA at pH 9.0 for heat-induced retrieval at 95–98 °C for 20 min (page retrieval setting). If staining is weak, compare a longer retrieval time on serial sections while holding antibody concentration and detection conditions constant; excessive heating can damage tissue morphology (standard IHC practice). Cool sections in retrieval buffer, then compare signal in the same cell populations and inspect tissue edges for stronger staining that could reflect uneven processing (standard IHC practice). The catalog image documents staining in mouse kidney at 2.5 µg/mL, but its caption does not report a fixative or retrieval method (A09744 caption).
Could fixation explain inconsistent OTUD5 staining between paraffin blocks?
OTUD5-specific sensitivity to fixation is unknown from the supplied evidence; the mouse kidney image caption does not state its fixative (A09744 caption). For paraffin IHC, compare blocks with documented fixation histories and process matched sections together before attributing a signal difference to biology (standard IHC practice). Keep section thickness, retrieval at pH 9.0 for 20 min, and antibody concentration constant across that comparison (page retrieval setting; standard IHC practice). If morphology is poor or staining concentrates at section edges, assess processing quality and repeat on a better-preserved block before scoring OTUD5 (standard IHC practice).
Should OTUD5 staining be nuclear or cytoplasmic in tissue sections?
Score nuclear and cytoplasmic staining separately: UniProt lists OTUD5 in the nucleus, while HPA reports general cytoplasmic tissue expression (UniProt Q96G74 subcellular location; HPA tissue IHC). HPA cell imaging places the main signal in cytosol, with additional nucleoplasmic and nucleolar fibrillar-center signal, so either compartment merits evaluation (HPA subcellular). On serial sections, compare compartment-specific signal with morphology and a no-primary control before calling diffuse color specific (standard IHC practice). If only one compartment stains, document that pattern instead of treating the other as obligatorily positive; HPA tissue IHC has medium consistency with RNA data and awaits external verification (HPA tissue IHC).
How could OTUD5 isoforms or epitope location affect IHC results?
OTUD5 has 5 listed isoforms and an OTU domain spanning residues 213–341; the supplied product caption does not identify the antibody epitope (UniProt Q96G74; A09744 caption). Without an epitope map, staining cannot establish which isoform is present or whether a particular domain is accessible after retrieval (UniProt Q96G74 isoforms and domains; standard IHC interpretation). OTUD5 also has documented modified residues, including phosphoserine at 64 and 165, but their effect on this antibody’s tissue staining is unknown (UniProt Q96G74 modified residues; A09744 caption). Compare an independently validated antibody with a known epitope on serial sections if isoform or epitope specificity is central to the experiment (standard IHC practice).
How can I check an OTUD5 IHC pattern with multiplex immunofluorescence?
Use IF on a matched section to examine compartment placement, pairing OTUD5 with a validated marker for the cell population being scored in IHC (standard IF practice). HPA reports mainly cytosolic signal with additional nucleoplasmic and nucleolar signal, so include a nuclear counterstain and assess each compartment separately (HPA subcellular; standard IF practice). OTUD5 has no transmembrane segment; choose permeabilisation for an intracellular epitope, then optimise it against morphology and background because this antibody’s epitope is unspecified (UniProt Q96G74 topology; A09744 caption; standard IF practice). Inspect unstained tissue for autofluorescence and place the OTUD5 channel in a spectrally separated, lower-background band before interpreting colocalisation (standard IF practice).
What should I change when OTUD5 chromogenic staining is diffuse?
First compare a no-primary section with the stained section to identify signal from detection reagents or endogenous activity (standard IHC practice). Include an appropriate peroxidase block before DAB detection and check whether residual color follows tissue structures rather than cells (standard chromogenic IHC practice). Titrate the primary antibody and shorten detection development if diffuse color rises across negative and positive regions; the mouse kidney caption reports 2.5 µg/mL but provides no general tissue dilution (A09744 caption; standard IHC practice). Check whether apparent cytoplasmic signal respects cell boundaries, because HPA describes general cytoplasmic tissue expression but rates its tissue evidence as approved with pending external verification (HPA tissue IHC).
How should I quantify OTUD5 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the compartment and cell population before scoring, then record percent positive cells and intensity or calculate an H-score from those observations (standard IHC scoring practice). Keep nuclear and cytoplasmic scores separate because the supplied sources report both nuclear location and cytoplasmic tissue staining (UniProt Q96G74 subcellular location; HPA tissue IHC). Normalise positive-cell counts to all evaluable cells of the same type, or stained area to evaluable tissue area in mm², excluding folds and necrosis by a prespecified rule (standard IHC scoring practice). Apply identical imaging, threshold and counterstain settings across sections, and compare matched regions because HPA reports low tissue RNA specificity (standard IHC practice; HPA tissue IHC).
How do I distinguish credible OTUD5 staining from artefact?
Interpret OTUD5 in identified cells and compartments, using the reported nuclear location and HPA’s mainly cytosolic cell signal as context rather than an absolute staining rule (UniProt Q96G74 subcellular location; HPA subcellular). HPA reports medium staining in appendix glandular cells and no detection in heart cardiomyocytes, but its tissue IHC remains pending external verification (HPA tissue IHC). Recheck a signal confined to unexpected cells or compartments against morphology, a no-primary control and an independently validated antibody when available (standard IHC practice). Exclude section-edge enhancement, necrotic regions and residual endogenous peroxidase color before calling a positive DAB result (standard chromogenic IHC practice).
Boster reagents
Best OTUD5 / OTU domain-containing protein 5 IHC Antibodies
Validated anti-OTUD5 antibodies have IHC data from mouse kidney tissue and IF data from human kidney cells and A431 cells (catalog image captions).
Real IHC data
Anti-OTUD5 Antibody
Cat # A09744
Real IF data
Anti-OTUD5 Antibody ®
Cat # A09744-1
A09744 supports IHC-P and IF, with an IHC image from mouse kidney tissue and an IF image from human kidney cells (catalog applications and image captions). A09744-1 supports ICC/IF, with an IF image from A431 cells (catalog applications and IF image caption).
Which to pick: Choose A09744 for paraffin-section tissue IHC: its application list includes IHC-P, and its own IHC image shows mouse kidney tissue at 2.5 μg/mL; the fixative is unreported (catalog applications; A09744 IHC image caption). Choose A09744-1 for ICC/IF of human cells: its application list includes ICC/IF, and its own image shows A431 cells at 5 μg/mL (catalog applications; A09744-1 IF image caption). For broader listed species reactivity, choose A09744, which lists human, mouse and rat; A09744-1 lists human and rat (catalog reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q96G74 (OTUD5_HUMAN, OTU domain-containing protein 5).
- Human Protein Atlas. OTUD5 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. OTUD5 subcellular location (ICC-IF): Mainly localized to the cytosol. In addition localized to the nucleoplasm and nucleoli fibrillar center..
- Human Protein Atlas. OTUD5 antibody validation summary (1 antibodies).
- Deubiquitinase OTUD5 modulates mTORC1 signaling to promote bladder cancer progression. Cell death & disease 2022 — PMC9463452.
- Hepatocyte Deubiquitinating Enzyme OTUD5 Deficiency is a Key Aggravator for Metabolic Dysfunction-Associated Steatohepatitis by Disturbing Mitochondrial Homeostasis. Cellular and molecular gastroenterology and hepatology 2024 — PMC10827517.
- Deubiquitinase OTUD5 promotes hepatitis B virus replication by removing K48-linked ubiquitination of HBV core/precore and upregulates HNF4ɑ expressions by inhibiting the ERK1/2/mitogen-activated protein kinase pathway. Cellular and molecular life sciences : CMLS 2023 — PMC10613150.
- OTUD5 maintains STAT1/2 stability and promotes IFNγ-driven intestinal inflammation. The Journal of biological chemistry 2026 — PMC13559827.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15772651 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for OTUD5 (OTU domain-containing protein 5). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC); nuclear location annotated (UniProt). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A09744 at 2.5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Epididymis. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet A09744); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-OTUD5, 2.5 μg/mL (datasheet A09744); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A09744); Caveat: Observed cytoplasmic staining differs from the nuclear annotation (HPA tissue IHC; UniProt); Isoform / epitope: Five isoforms; epitope coverage is unresolved (UniProt); Regulation: Expression regulation is not established (UniProt). Evidence: UniProt Q96G74, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the cytosol. In addition localized to the…, Datasheet A09744, PMC9463452, PMC10613150. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/OTUD5/immunohistochemistry-ihc-guide.