This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
USP9X / Ubiquitin carboxyl-terminal hydrolase 9X · IHC design guide
Design Immunohistochemistry for USP9X
Plan USP9X paraffin IHC using the catalog antibody at 1:50 as a starting dilution (datasheet M02594-1). Assess granular cytoplasmic staining across tissues and account for cell types reported as undetected (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
USP9X 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 | Granular cytoplasmic staining in tissue (HPA tissue IHC) | |
| Staining pattern | Granular cytoplasm across tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M02594-1) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Heart muscle+1 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Cardiomyocytes and smooth muscle cells may lack signal (HPA tissue IHC) | |
| Regulation | Widely expressed in embryonic and adult tissues (UniProt) | |
| Isoform / epitope | 2 isoforms; verify antibody epitope coverage (UniProt) |
Section 1
Recommended USP9X IHC & IF Protocols
The catalog antibody’s IHC-P protocol is paired with four published USP9X IHC protocols for comparing sample preparation, staining, and detection (datasheet M02594-1; PMC13346229; PMC6568031; PMC4016599; PMC5346732).
| Sample | Paraffin-embedded human testis cancer tissue; fixative not specified (datasheet M02594-1) |
| Fixation | Image fixative and duration unreported (datasheet M02594-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet M02594-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M02594-1) |
| Primary antibody | Rabbit monoclonal (clone 18U88) anti-USP9X, 1:50 (datasheet M02594-1) |
| Primary incubation | Overnight at 4 °C (datasheet M02594-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet M02594-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | USP9X-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Granular cytoplasmic expression in all tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet M02594-1); the esophageal study reports boiling citrate at pH 6.0 as a published alternative (PMC4016599).
Section 2
What Is the Expected USP9X Staining Pattern?
USP9X should show granular cytoplasmic staining across many tissues, with strong signal in selected glandular, hematopoietic, neuronal and squamous epithelial cells (HPA: tissue IHC). Its reported locations include cytosol, growth cones and cilium axonemes; it has no transmembrane segment (UniProt Q93008: location and topology). HPA rates the tissue IHC profile Approved, with medium agreement between staining and RNA data and external verification pending (HPA: reliability).
| Granular cytoplasmic signal in colon or duodenal glandular cells, marrow hematopoietic cells, or cortical neurons. | This matches reported high staining in these cells (HPA: tissue IHC). Judge the pattern within the identified cell population and alongside tissue controls; signal intensity alone does not establish antibody specificity (general IHC practice). |
| Predominantly nuclear, crisp plasma membrane, or extracellular staining, with little cytoplasmic signal. | This conflicts with the reported granular cytoplasmic IHC pattern and supported cytosolic ICC-IF location (HPA: tissue IHC and subcellular). Check morphology and controls before calling it USP9X; an isolated wrong-compartment pattern may reflect artefact (general IHC practice). |
| Strong signal in cardiomyocytes or smooth muscle cells while expected positive cells are weak. | HPA reports USP9X as not detected in those cell types (HPA: tissue IHC). Consider cross-reactivity or endogenous detection activity, and compare with a reagent-omission control; HPA’s Approved profile still awaits external verification (HPA: reliability; general IHC practice). |
| Uniform colour over cells, stroma and empty areas, obscuring cell boundaries. | That distribution is inconsistent with a cell-associated granular cytoplasmic pattern (HPA: tissue IHC). Diffuse background can arise from nonspecific binding, incomplete washing or detection chemistry; inspect a negative reagent control before scoring cells (general IHC practice). |
| No signal in a well-preserved section of a reported high-staining tissue. | First confirm that the expected cell population is present: HPA reports high staining in adrenal glandular cells, for example (HPA: tissue IHC). Then review section processing, antigen retrieval, reagent performance and detection controls as general IHC checks; this record does not establish USP9X-specific fixation sensitivity. |
💡Expected USP9X appearanceCall a positive result when the relevant cells show discernible granular cytoplasmic staining, often high in HPA-listed positive populations (HPA: tissue IHC); dominant nuclear, membrane or structure-free colour warrants review against controls (HPA: location; general IHC practice).
| Tissue and cell population | Expression is broad but intensity varies: HPA lists high staining in several glandular populations and neurons, low staining in prostate glandular cells, and no detected staining in cardiomyocytes (HPA: tissue IHC). Choose and score controls by cell type rather than whole-section colour (general IHC practice). |
| Subcellular location and topology | UniProt places USP9X in cytosol, growth cones and cilium axonemes and records no transmembrane segment (UniProt Q93008: location and topology). HPA describes the tissue signal as granular cytoplasmic; neither source supports treating a membrane-only IHC pattern as expected (HPA: tissue IHC). |
| Antibody evidence | CAB011618 and CAB070164 have Approved IHC status (HPA: antibodies). The tissue profile has medium agreement with RNA data and awaits external verification (HPA: reliability), so controls remain necessary when a result departs from the reported pattern (general IHC practice). |
| Isoforms and epitope interpretation | UniProt lists 2 isoforms and a full-length chain spanning residues 1–2554 (UniProt Q93008: isoforms and processing). The supplied sources do not map either antibody’s epitope or show isoform-specific IHC staining; do not assign a tissue pattern to one isoform. |
| IF/ICC: should it look similar? | Yes, cytosolic signal is supported in ICC-IF, and CAB011618 has Supported ICC status (HPA: subcellular and antibodies). Compare compartment-level findings cautiously because this section concerns chromogenic IHC in paraffin tissue, while HPA’s ICC-IF evidence comes from cell images. |
| Situation | Likely cause | Next action |
|---|---|---|
| Reported positive glandular cells show no staining. | The expected cells may be absent, or a step in section processing, retrieval or detection may have failed (HPA: high glandular-cell staining; general IHC practice). | Verify cell identity and preservation, then check the catalog antibody’s IHC-P instructions and run appropriate positive and detection controls (general IHC practice). |
| Signal is predominantly nuclear or at the cell surface. | The compartment disagrees with granular cytoplasmic tissue staining and cytosolic ICC-IF localisation (HPA: tissue IHC and subcellular). | Review morphology, staining distribution and reagent-omission controls; interpret the discordant signal cautiously until specificity is supported (general IHC practice). |
| Cardiomyocytes or smooth muscle cells stain strongly. | These cells are reported as not detected by HPA; cross-reactivity or endogenous detection activity is possible (HPA: tissue IHC; general IHC practice). | Check a reagent-omission control and compare the same run with a cell population reported to stain strongly (HPA: tissue IHC; general IHC practice). |
| Background covers stroma or blank spaces. | The spread of colour differs from cell-associated granular staining; nonspecific binding, washing or detection chemistry may contribute (HPA: tissue IHC; general IHC practice). | Inspect controls, blocking and washes, then adjust general IHC conditions as indicated by the control result (general IHC practice). |
| Signal is weak in prostate glandular cells or ovarian stroma. | Low staining is reported for these populations, so a faint result can agree with the reference pattern (HPA: tissue IHC). | Use a reported high-staining population to assess run performance before changing conditions; score the low-staining cells separately (HPA: tissue IHC; general IHC practice). |
| Two sections differ in staining intensity. | Differences in sampled cell populations can matter because HPA reports both high and low staining across tissues (HPA: tissue IHC). The supplied sources do not establish a USP9X-specific fixation effect. | Compare like cell populations and same-run controls, then review routine processing and detection records if the difference persists (general IHC practice). |
Experiment evidence
IHC & IF images showing USP9X staining
M02594-1 · IHCIHC analysis of USP9X using anti-USP9X antibody (M02594-1). USP9X was detected in a paraffin-embedded section of human testis cancer tissue.
M02594-1 · IHCIHC analysis of USP9X using anti-USP9X antibody (M02594-1). USP9X was detected in a paraffin-embedded section of mouse liver tissue. Heat me
A02594-1 · IFIF analysis of USP9X using anti-USP9X antibody (A02594-1). USP9X was detected in an immunocytochemical section of A549 cells. Enzyme antigen
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000124486-USP9X/tissue/cerebral+cortex#imid_3069000
HPA · Parathyroid glandHuman Protein Atlas IHC, Parathyroid gland. Source: https://www.proteinatlas.org/ENSG00000124486-USP9X/tissue/parathyroid+gland#imid_3068899
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000124486-USP9X/tissue/colon#imid_3068926
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000124486-USP9X/tissue/liver#imid_3068935
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB011618; green = USP9X, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB011618; green = USP9X, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for USP9X IHC & IF
🧪Run adrenal gland first: glandular cells should stain (HPA: High in adrenal gland glandular cells). Run heart muscle as the negative tissue: cardiomyocytes are not detected (HPA: Not detected in heart muscle cardiomyocytes); on the adrenal slide, use non-glandular areas to assess background, without treating them as a validated USP9X-negative cell population.
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Heart muscle (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show USP9X in A-431, U-251MG, U2OS, with annotated localisation: Cytosol (supported) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) slide, a concentration-matched rabbit IgG isotype control, and a matched USP9X-knockout biological control; the selected antibody is rabbit anti-USP9X (selected-SKU IHC caption). Quench endogenous peroxidase and check for background in the blood-rich adrenal section before interpreting HRP/DAB staining (standard IHC practice; selected-SKU IHC caption: HRP/DAB detection).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU paraffin-section caption does not state its fixative (selected-SKU IHC caption: fixative unreported). Heat retrieval in EDTA at pH 8.0 was used for the caption’s testis cancer section, but retrieval dependence in adrenal gland is unreported (selected-SKU IHC caption). The supplied evidence does not establish whether frozen sections or IF are easier; USP9X has supported cytosolic localization in ICC-IF images, while endogenous peroxidase in a blood-rich adrenal section can complicate HRP/DAB interpretation (HPA: Cytosol supported; standard IHC practice).
HPA tissue IHC evidence for USP9X
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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
Section 3
Advanced USP9X IHC Tips
Troubleshoot USP9X staining in paraffin sections using the catalog antibody’s tissue image and reported cytoplasmic localisation (datasheet M02594-1; HPA tissue IHC).
Which retrieval condition should I use when USP9X staining is weak in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for the catalog antibody (datasheet M02594-1). The reported paraffin-section image used that condition, 1:50 primary antibody overnight at 4°C, and DAB detection (datasheet M02594-1). If staining remains weak, adjust heating duration and cooling consistently across a small set of adjacent sections, then compare cytoplasmic signal with tissue damage and background (standard IHC practice; HPA tissue IHC). Keep the antibody concentration and detection steps fixed during that comparison so retrieval is the variable being assessed (standard IHC practice). Record the selected condition for every batch, because inconsistent retrieval can mimic a change in USP9X expression (standard IHC practice).
How should I troubleshoot variable USP9X staining when fixation histories differ?
Target-specific sensitivity of USP9X staining to fixation is unknown from the supplied evidence; the catalog tissue caption describes a paraffin section but does not state its fixative (datasheet M02594-1). Record the available fixation and processing history for each specimen before comparing staining intensity (standard IHC practice). Compare sections processed together with the same EDTA pH 8.0 retrieval and detection settings, using internal tissue morphology to flag poorly preserved regions (datasheet M02594-1; standard IHC practice). If histories differ, report that limitation alongside any intensity difference instead of assigning it to USP9X biology (standard IHC practice). Do not infer fixation sensitivity from cytoplasmic localisation or phosphorylation sites (UniProt Q93008; standard IHC practice).
What staining compartment should I expect, and how should I assess unexpected nuclear signal?
Look first for granular cytoplasmic staining in tissue sections, consistent with the reported tissue profile and supported cytosolic localisation (HPA tissue IHC; HPA subcellular). USP9X is also annotated at growth cones and the cilium axoneme, so cell context matters when reviewing a localised projection signal (UniProt Q93008). For predominantly nuclear staining, compare adjacent sections, inspect the counterstain and morphology, and check whether the same pattern appears without primary antibody (standard IHC practice). Score cytoplasmic and nuclear signal separately rather than combining them into one intensity value (standard IHC practice). Treat a new compartment pattern as provisional until a second specificity check supports it (standard IHC practice).
Can this antibody distinguish USP9X isoforms or altered epitopes in paraffin sections?
The record lists 2 USP9X isoforms, but the supplied catalog caption does not map this antibody’s epitope or establish isoform selectivity (UniProt Q93008; datasheet M02594-1). USP9X has no annotated transmembrane segment, while its USP domain spans residues 1557–1956 (UniProt Q93008). Its annotated phosphorylation sites include residues 588, 590, 1600, 2443, and 2540; their effects on this antibody’s staining are unreported (UniProt Q93008; datasheet M02594-1). If staining differs across specimens, verify the epitope and isoform coverage from antibody documentation before assigning the difference to splicing or modification (standard IHC practice). Record retrieval conditions alongside that assessment, since epitope accessibility can affect IHC comparisons (standard IHC practice).
How can IF help investigate an ambiguous USP9X IHC pattern?
Use IF as a complementary localisation check: supported cytosolic localisation and granular cytoplasmic tissue staining provide the expected pattern to examine (HPA subcellular; HPA tissue IHC). Multiplex USP9X with a validated marker for the cell population being assessed, and include single-stain controls to check channel bleed-through (standard IF practice). Choose fluorophores after inspecting tissue autofluorescence in each channel, with particular care when weak USP9X signal overlaps the background spectrum (standard IF practice). Because USP9X has no transmembrane segment and is primarily cytosolic, assess permeabilisation that permits intracellular antibody access without losing cell structure (UniProt Q93008; standard IF practice). Do not carry the paraffin-section retrieval setting into IF without testing it for the IF specimen preparation (datasheet M02594-1; standard IF practice).
How do I separate weak USP9X signal from chromogenic background?
The catalog paraffin-section example used 10% goat serum blocking, a peroxidase-conjugated secondary antibody, and DAB chromogen (datasheet M02594-1). When diffuse brown staining obscures cytoplasm, inspect a section without primary antibody and review endogenous peroxidase blocking as part of the chromogenic workflow (standard IHC practice). Titrate primary antibody around the reported 1:50 condition while keeping retrieval, DAB development, and imaging settings constant (datasheet M02594-1; standard IHC practice). Compare signal within intact cells against nearby stromal and acellular areas rather than judging overall section colour (standard IHC practice). A patterned cytoplasmic signal is more consistent with the reported USP9X tissue profile than uniform edge or extracellular deposit staining (HPA tissue IHC; standard IHC practice).
How should I score USP9X staining across tissue samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and cytoplasmic scoring region before reviewing groups, since the reported tissue profile is granular and cytoplasmic (HPA tissue IHC; standard IHC practice). For cell-level staining, record percent positive cells and an intensity grade, then calculate a cytoplasmic H-score using the same thresholds across samples (standard IHC practice). For spatial questions, count positive cells per mm² of viable tissue and report the analysed area (standard IHC practice). Normalise comparisons to the same cell type, viable tissue area, section handling, retrieval, and detection batch where possible (standard IHC practice). Exclude necrosis, folded tissue, and edge artefacts using prespecified rules, and keep representative images for review (standard IHC practice).
When should an apparent USP9X-positive region be treated as artefact?
Assess whether the signal lies in intact cell cytoplasm, because USP9X is supported in the cytosol and tissue staining is described as granular cytoplasmic (HPA subcellular; HPA tissue IHC). High staining has been reported in cerebral-cortex neuronal cells and several glandular-cell populations, while cardiomyocytes and smooth-muscle cells were reported as not detected (HPA tissue IHC). Inspect unexpected cell or nuclear staining against morphology and a section without primary antibody before calling it positive (standard IHC practice). Edge effects, necrotic deposits, and residual endogenous peroxidase can produce misleading DAB signal, so exclude affected regions from scoring (standard IHC practice). Interpret small differences cautiously because the tissue IHC assessment has medium staining–RNA consistency and awaits external verification (HPA tissue IHC).
Boster reagents
Best USP9X / Ubiquitin carboxyl-terminal hydrolase 9X IHC Antibodies
Anti-USP9X antibodies have IHC images from paraffin sections of human testis cancer and mouse liver (M02594-1 IHC captions), and an IF image from A549 cells (A02594-1 IF caption).
Real IHC data
Anti-USP9x Rabbit Monoclonal Antibody
Cat # M02594-1
Real IF data
Anti-USP9X Antibody ®
Cat # A02594-1
M02594-1 lists IHC and has images from paraffin sections of human testis cancer and mouse liver (catalog applications; M02594-1 IHC captions). A02594-1 lists IF/ICC and has an IF image from A549 cells (catalog applications; A02594-1 IF caption).
Which to pick: For tissue IHC, choose M02594-1: it is a rabbit monoclonal with paraffin-section IHC images; the fixative is unreported (catalog host/clone; M02594-1 IHC captions). For IF/ICC, choose A02594-1 for its A549 IF image at 5 μg/mL (A02594-1 IF caption). Both list human, mouse and rat reactivity, while the tissue IHC images show human and mouse samples (catalog reactivity; M02594-1 IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q93008 (USP9X_HUMAN, Ubiquitin carboxyl-terminal hydrolase 9X).
- Human Protein Atlas. USP9X tissue IHC expression (reliability: Approved).
- Human Protein Atlas. USP9X subcellular location (ICC-IF): Localized to the cytosol..
- Human Protein Atlas. USP9X antibody validation summary (2 antibodies).
- Bioinformatics screening identifies USP9X as a pathogenic gene underlying thyroid and breast cancer comorbidity. Journal of molecular histology 2026 — PMC13346229.
- Overexpression of the Ubiquitin-Specific Peptidase 9 X-Linked (USP9X) Gene is Associated with Upregulation of Cyclin D1 (CCND1) and Downregulation of Cyclin-Dependent Inhibitor Kinase 1A (CDKN1A) in Breast Cancer Tissue and Cell Lines. Medical science monitor : international medical journal of experimental and clinical research 2019 — PMC6568031.
- USP9X expression correlates with tumor progression and poor prognosis in esophageal squamous cell carcinoma. Diagnostic pathology 2013 — PMC4016599.
- Deubiquitinase USP9X deubiquitinates β-catenin and promotes high grade glioma cell growth. Oncotarget 2016 — PMC5346732.
- PubMed PMID:8922996 — UniProt-cited evidence.
- PubMed PMID:15772651 — UniProt-cited evidence.
- PubMed PMID:15592455 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for USP9X (Ubiquitin carboxyl-terminal hydrolase 9X). Expected localisation Granular cytoplasmic staining in tissue (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M02594-1). Validated IHC antibody: M02594-1 at 1:50 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Heart muscle. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human testis cancer tissue; fixative not specified (datasheet M02594-1); 2. Fixation: Image fixative and duration unreported (datasheet M02594-1); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: EDTA pH 8.0 (datasheet M02594-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M02594-1); 8. Primary antibody: Rabbit monoclonal (clone 18U88) anti-USP9X, 1:50 (datasheet M02594-1); 9. Primary incubation: Overnight at 4 °C (datasheet M02594-1). Watch for: Fixation: Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific); Caveat: Cardiomyocytes and smooth muscle cells may lack signal (HPA tissue IHC); Isoform / epitope: 2 isoforms; verify antibody epitope coverage (UniProt); Regulation: Widely expressed in embryonic and adult tissues (UniProt). Evidence: UniProt Q93008, HPA tissue IHC · Approved, HPA ICC-IF · cytosol., Datasheet M02594-1, PMC13346229, PMC6568031. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/USP9X/immunohistochemistry-ihc-guide.