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USP9X / Ubiquitin carboxyl-terminal hydrolase 9X · IHC design guide
Design Immunohistochemistry for USP9X
This guide uses the IHC-validated antibody at 1:50 for chromogenic staining of paraffin sections (datasheet M02594-1). Expect granular cytoplasmic signal in positive cells, with colon glandular cells as a high-staining reference and heart cardiomyocytes as an undetected reference (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 (HPA tissue IHC) | |
| Staining pattern | Granular cytoplasm in positive tissue cells (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 consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Cardiomyocytes may lack staining (HPA tissue IHC) | |
| Regulation | Expression regulation is unspecified (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope effects are unknown (UniProt) |
Section 1
Recommended USP9X IHC & IF Protocols
The catalog antibody’s IHC protocol is followed by published USP9X staining conditions for breast, esophageal and glioma tissue (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 at pH 8.0 for the catalog antibody (datasheet M02594-1). Use a paper’s stated retrieval conditions when reproducing its assay (PMC6568031; PMC4016599; PMC5346732).
Section 2
What Is the Expected USP9X Staining Pattern?
USP9X should appear mainly as granular cytoplasmic staining in tissue sections (HPA: tissue IHC profile). Cytosolic localisation is consistent with a protein lacking a transmembrane segment (UniProt Q93008: localisation and topology). HPA reports high staining in colon glandular cells, cerebral cortex neurons and bone marrow hematopoietic cells, among others (HPA: tissue IHC). Its IHC assessment is Approved, with medium consistency between staining and RNA data and external verification pending (HPA: reliability).
| Granular cytoplasmic staining in colon glandular cells or cerebral cortex neurons. | This matches the reported compartment and high-staining cell populations (HPA: tissue IHC). Judge the relevant cells individually; a tissue-level positive result does not mean every cell must stain equally (general IHC practice). |
| Predominantly nuclear, sharply membranous or extracellular staining. | These are unexpected as the dominant pattern: HPA describes granular cytoplasm, while UniProt lists cytosol and no transmembrane segment (HPA: tissue IHC; UniProt Q93008: localisation and topology). Check whether the signal survives appropriate controls before interpreting it as USP9X (general IHC practice). |
| Strong staining in cardiomyocytes or smooth muscle cells. | HPA reports USP9X as not detected in those cell populations (HPA: heart muscle and smooth muscle IHC). Consider cross-reactivity or detection-system activity, and compare with a known-positive population on the same run (general IHC practice). |
| Diffuse colour across cells, stroma and slide background. | That distribution does not resemble HPA's granular cytoplasmic profile (HPA: tissue IHC). Uneven blocking, residual detection activity or excess reagent can cause background; inspect a no-primary control and the counterstain before scoring (general IHC practice). |
| No signal in a section expected to contain high-staining cells. | First confirm that the relevant cells are present and preserved, such as colon glandular cells (HPA: high in colon glandular cells). If they are, review the IHC-validated antibody, antigen-retrieval conditions and detection controls; HPA supplies no USP9X-specific retrieval requirement (HPA: antibody IHC status; general IHC practice). |
💡Expected USP9X appearanceA convincing positive is granular cytoplasmic signal in expected cells, potentially strong in colon glandular cells or cortical neurons (HPA: tissue IHC); dominant nuclear or membranous colour, or strong cardiomyocyte staining, warrants a false-positive check (HPA: tissue IHC; UniProt Q93008: topology).
| Cell compartment and tissue choice (HPA: tissue IHC; UniProt Q93008: localisation). | Use a reported high-staining population, such as appendix glandular cells or bone marrow hematopoietic cells, to assess the cytoplasmic pattern (HPA: tissue IHC). UniProt also lists growth cones and cilium axonemes; their listing does not establish a distinctive paraffin-section pattern (UniProt Q93008: localisation). |
| IHC antibody evidence (HPA: antibody validation). | CAB011618 and CAB070164 are both IHC Approved (HPA: antibody validation). Approved here means medium consistency with RNA and pending external verification, so unexpected staining needs controls; neither antibody is listed as IHC Enhanced in the supplied record (HPA: reliability and antibody validation). |
| Expression range and low-signal populations (HPA: tissue IHC). | HPA describes expression in all tissues but lists low staining in prostate glandular cells, ovarian stroma, skeletal myocytes and adipocytes, with no detection in cardiomyocytes and smooth muscle cells (HPA: tissue IHC). Treat these as cell-population observations, not universal tissue negatives (HPA: tissue IHC). |
| Molecular features and interpretation limits (UniProt Q93008: isoforms, processing and modifications). | UniProt lists two isoforms, one full-length chain, no signal peptide and several modified residues (UniProt Q93008). The payload gives no antibody epitope or evidence that these features alter paraffin staining; do not assign an unexplained result to an isoform or modification. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive glandular cells look blank (HPA: high in colon glandular cells). | The cells may be absent from the section, or the IHC workflow may have failed (general IHC practice). | Verify morphology, then compare a known-positive section and run controls; review retrieval, primary-antibody use and detection in that order (general IHC practice). No USP9X-specific retrieval condition is supplied. |
| Colour appears mainly in nuclei or at cell borders. | That dominant distribution conflicts with the reported cytoplasmic pattern (HPA: tissue IHC; UniProt Q93008: localisation). | Compare with a no-primary control and the expected cytoplasmic signal; repeat with an IHC Approved antibody if the mismatch persists (HPA: antibody validation; general IHC practice). |
| Strong signal appears in cardiomyocytes or smooth muscle cells. | These populations are reported as not detected; cross-reactivity or endogenous detection activity is possible (HPA: tissue IHC; general IHC practice). | Check a no-primary control, block endogenous detection activity as appropriate, and compare an expected high-staining population (HPA: tissue IHC; general IHC practice). |
| Background obscures cytoplasmic granules. | Nonspecific reagent binding or detection background may obscure the reported pattern (HPA: tissue IHC; general IHC practice). | Inspect no-primary and detection-only controls; optimise blocking, washes and primary-antibody concentration using the antibody's IHC instructions (general IHC practice). |
| A weak result is judged negative in prostate glandular cells or adipocytes. | HPA assigns those populations low staining, so faint signal can be consistent with its observations (HPA: tissue IHC). | Use a reported high-staining population for the run control and score the low-staining population against its local background (HPA: tissue IHC; general IHC practice). |
| Will IF/ICC show the same compartment? | HPA reports supported cytosolic ICC-IF localisation; CAB011618 has ICC Supported status (HPA: subcellular summary and antibody validation). | Expect cytosolic fluorescence as a localisation check (HPA: ICC-IF). Use the separate IF/ICC guide for that assay; tissue IHC intensity does not specify an IF setting (HPA: tissue IHC and ICC-IF). |
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 and expect staining in glandular cells (HPA: High in adrenal gland glandular cells); use heart muscle as the negative tissue and expect cardiomyocytes to remain unstained (HPA: Not detected in heart muscle cardiomyocytes). On the adrenal slide, assess cells outside the glandular compartment for absent staining, but do not designate them confirmed internal negatives because their USP9X status is not reported in the supplied HPA row (HPA: adrenal gland glandular cells only).
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 control, a concentration-matched rabbit IgG isotype control, and USP9X-knockout material as a biological negative (caption: rabbit anti-USP9X primary; standard IHC practice). Quench endogenous peroxidase and inspect the adrenal slide for pigment that could be mistaken for DAB signal (caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected paraffin-section caption does not state the fixative (caption: fixative not stated). The reported IHC procedure used heat retrieval in EDTA at pH 8.0, so that is a documented starting condition; a requirement for retrieval across specimens is unreported (caption: EDTA pH 8.0). The supplied evidence does not establish that frozen sections or IF are easier; assess adrenal pigment when interpreting chromogenic staining (HPA: adrenal gland positive; caption: DAB detection; 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 chromogenic IHC by checking retrieval, cytoplasmic localisation, controls and cell-level scoring (datasheet M02594-1; HPA tissue IHC).
What retrieval should I try 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 demonstrated paraffin-section workflow then used 10% goat serum and primary antibody at 1:50 overnight at 4°C (datasheet M02594-1). If staining remains weak, compare retrieval heating and cooling conditions on matched sections while keeping antibody concentration and detection constant (standard IHC practice). Inspect morphology alongside signal, since excessive retrieval can damage tissue and make cytoplasmic staining difficult to assign to cells (standard IHC practice). Record each retrieval condition with its control section before selecting one for scoring (standard IHC practice).
How should I troubleshoot variable USP9X staining after tissue fixation?
Target-specific USP9X sensitivity to fixative type or fixation duration is unknown from the supplied evidence (datasheet M02594-1). The catalog image describes a paraffin-embedded section but does not state its fixative, so it cannot establish a formalin fixation condition (datasheet M02594-1). Record fixative, fixation duration, processing history and section age for each specimen before comparing signal (standard IHC practice). When material permits, stain matched sections together using the same EDTA pH 8.0 retrieval and antibody conditions to isolate processing differences (datasheet M02594-1; standard IHC practice). Interpret changes against morphology and a positive control, without assigning a USP9X-specific fixation effect from staining alone (standard IHC practice).
Where should convincing USP9X staining appear within a positive cell?
Expect predominantly granular cytoplasmic staining in tissue sections, with cytosolic localisation supported by subcellular imaging (HPA tissue IHC; HPA subcellular). USP9X is also annotated at growth cones and the cilium axoneme, so specialised projections may merit closer inspection when present (UniProt Q93008 subcellular). It has no transmembrane segment, making a crisp membrane-only pattern an unexpected result for this target (UniProt Q93008 topology). Check whether DAB lies within intact cell borders rather than over folds, detached tissue or extracellular debris (standard IHC practice). Compare suspicious patterns with the counterstain and a reagent control before calling them positive (standard IHC practice).
Could isoforms or epitope accessibility explain a discordant staining pattern?
USP9X has 2 annotated isoforms, but the supplied catalog caption does not identify this antibody’s epitope or isoform coverage (UniProt Q93008 isoforms; datasheet M02594-1). Its USP domain spans residues 1557–1956, and annotated phosphorylation sites include residues 588, 590, 1600, 2443 and 2540 (UniProt Q93008 domains and modified residues). Those annotations alone cannot establish that a particular isoform or modification changes this antibody’s IHC signal (UniProt Q93008; datasheet M02594-1). Obtain the immunogen or epitope information before making an isoform claim, and compare a second antibody only after checking its stated specificity (standard IHC validation practice). Keep retrieval and detection matched when comparing antibodies, so technical differences remain interpretable (standard IHC practice).
How can I use IF to investigate ambiguous USP9X IHC localisation?
Use IF as a localisation cross-check: cytosol is the supported subcellular location, while the selected catalog evidence here describes chromogenic IHC (HPA subcellular; datasheet M02594-1). Multiplex USP9X with a validated marker for the cell type being assessed, such as a neuronal marker when examining cerebral cortex neuronal cells (HPA tissue IHC; standard IF practice). Choose a fluorophore channel with low tissue autofluorescence, checking an unstained section before interpreting weak signal (standard IF practice). Because USP9X lacks a transmembrane segment and is annotated in the cytosol, assess permeabilisation appropriate for intracellular access; the antibody’s precise epitope is unspecified (UniProt Q93008 topology and subcellular; datasheet M02594-1). Keep single-label controls to assess bleed-through in the multiplex image (standard IF practice).
What controls help distinguish USP9X staining from diffuse DAB background?
The documented assay used a peroxidase-conjugated secondary antibody and DAB, so endogenous peroxidase activity is a relevant background check (datasheet M02594-1; standard IHC practice). Include a peroxidase block and a section processed without primary antibody when investigating widespread brown deposits (standard IHC practice). The catalog workflow used 10% goat serum before primary antibody at 1:50 overnight at 4°C (datasheet M02594-1). If background persists, compare antibody dilution, washing and detection time on adjacent sections while retaining a positive control (standard IHC practice). Score only staining assigned to intact cellular cytoplasm, consistent with the reported tissue pattern (HPA tissue IHC; standard IHC practice).
How should I quantify USP9X IHC across specimens with different cellularity? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored compartment as cytoplasm before analysis, consistent with the reported granular tissue pattern (HPA tissue IHC). For comparable cell populations, record the percentage of positive cells and staining intensity, then calculate an H-score from intensity categories 0–3 if that scale is prespecified (standard IHC scoring practice). Normalise positive-cell counts to the number of eligible cells in each region, or report positive-cell density per mm² with the sampled area stated (standard IHC scoring practice). Exclude necrosis, folds and damaged edges using the same rules across sections (standard IHC practice). Because baseline expression spans many tissues and cell types, compare like cell populations and matched processing batches (UniProt Q93008 tissue specificity; HPA tissue IHC; standard IHC practice).
When is an apparent USP9X-positive region likely to be artefact?
A credible positive shows cytoplasmic signal within intact cells, matching the reported granular tissue pattern and supported cytosolic location (HPA tissue IHC; HPA subcellular). Treat isolated nuclear or membrane-only staining as questionable until confirmed, since the supplied localisation evidence supports cytosol and USP9X has no transmembrane segment (HPA subcellular; UniProt Q93008 topology). Verify cell identity: HPA reports high staining in cerebral cortex neuronal cells, whereas heart muscle cardiomyocytes were not detected in its tissue panel (HPA tissue IHC). Signal restricted to section edges, necrotic areas or tissue folds requires control review (standard IHC practice). Residual DAB in a no-primary control points to detection background, including possible endogenous peroxidase, rather than target staining (standard IHC practice).
Boster reagents
Best USP9X / Ubiquitin carboxyl-terminal hydrolase 9X IHC Antibodies
Anti-USP9X antibodies have IHC images from human testis cancer and mouse liver paraffin sections (M02594-1 image captions), plus an IF image from A549 cells (A02594-1 image caption).
Real IHC data
Anti-USP9x Rabbit Monoclonal Antibody
Cat # M02594-1
Real IF data
Anti-USP9X Antibody ®
Cat # A02594-1
M02594-1 is listed for IHC and IF/ICC (catalog applications), with IHC images from human testis cancer and mouse liver paraffin sections (M02594-1 image captions). A02594-1 is listed for IF/ICC (catalog applications), with an IF image from A549 cells (A02594-1 image caption).
Which to pick: For tissue IHC, choose M02594-1: it is a rabbit monoclonal listed for IHC (catalog), and its images show paraffin sections of human testis cancer and mouse liver (M02594-1 image captions); the fixative is unreported (M02594-1 image captions). For IF/ICC, choose A02594-1 for its A549 cell IF image (A02594-1 image caption) and listed IF/ICC applications (catalog). For cross-species planning, both SKUs list human, mouse and rat reactivity (catalog); direct tissue IHC images are available for human and mouse with M02594-1 (M02594-1 image 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 (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 consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Cardiomyocytes may lack staining (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope effects are unknown (UniProt); Regulation: Expression regulation is unspecified (UniProt). Evidence: UniProt Q93008, HPA tissue IHC · Approved, HPA ICC-IF · cytosol., Datasheet M02594-1, PMC6568031, PMC4016599. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/USP9X/immunohistochemistry-ihc-guide.