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USP5 / Ubiquitin carboxyl-terminal hydrolase 5 · IHC design guide
Design Immunohistochemistry for USP5
Plan USP5 chromogenic IHC in paraffin sections using the catalog antibody’s tissue protocol and a testis control (datasheet A04550-1; HPA tissue IHC). Compare cytoplasmic and nuclear staining with the reported tissue pattern, while accounting for its uncertain reliability (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
USP5 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 and nuclear staining in several tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic and nuclear; high in seminiferous duct cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A04550-1) | |
| Positive control | Cerebellum+4 more · see all | |
| Negative control | Salivary gland+2 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 A04550-1) | |
| Caveat | Low staining–RNA concordance; validate the tissue pattern (HPA tissue IHC) | |
| Regulation | No abundance regulator annotated (UniProt) | |
| Isoform / epitope | 2 isoforms (Long, Short); epitope coverage is unknown (UniProt) |
Section 1
Recommended USP5 IHC & IF Protocols
The catalog antibody protocol uses EDTA retrieval (datasheet A04550-1). The published USP5 IHC protocols below describe colorectal cancer (PMC6592179), human tumor tissue (PMC6991651), and NSCLC (PMC9826599).
| Sample | Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A04550-1) |
| Fixation | Image fixative and duration unreported (datasheet A04550-1); 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 A04550-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A04550-1) |
| Primary antibody | Rabbit anti-USP5, 2-5 μg/ml (datasheet A04550-1) |
| Primary incubation | Overnight at 4 °C (datasheet A04550-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A04550-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | USP5-positive staining in purkinje cells of cerebellum (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and nuclear expression in several tissues, including testis, cerebral cortex and skin. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet A04550-1). A published USP5 IHC protocol used citrate pH 6.0 pressure cooker retrieval (PMC9826599).
Section 2
What Is the Expected USP5 Staining Pattern?
USP5 should show cytoplasmic and nuclear staining in several tissues, with strong staining reported in cerebellar Purkinje cells and cells in testicular seminiferous ducts (HPA: tissue IHC). A membrane-bound pattern is not expected (UniProt P45974: cytoplasm, nucleus; no transmembrane segment). Interpret tissue results cautiously: HPA rates its IHC evidence Uncertain because antibody staining has low consistency with RNA expression (HPA: IHC reliability).
| Cytoplasmic and nuclear staining in Purkinje cells or cells in seminiferous ducts. | This matches HPA's High staining calls in those cell populations and its reported cytoplasmic and nuclear tissue pattern (HPA: tissue IHC). Compare compartments within identifiable cells; the HPA reliability rating remains Uncertain, so a matching image alone does not establish antibody specificity (HPA: IHC reliability). |
| Signal restricted to cell membranes or extracellular spaces, without convincing cytoplasmic or nuclear staining. | That distribution conflicts with the reported locations and lack of a transmembrane segment (UniProt P45974: subcellular location, topology; HPA: tissue IHC). Treat it as a possible staining artefact and review morphology, controls and detection conditions before assigning it to USP5 (general IHC practice). |
| Strong staining in salivary glandular cells, smooth muscle cells or soft tissue fibroblasts. | HPA reports USP5 as Not detected in those specific cell populations (HPA: tissue IHC). Unexpected signal warrants a check for cross-reactivity or endogenous detection activity (general IHC practice). These HPA observations are comparison points, not proof that every cell in those tissues must be negative (HPA: tissue IHC, Uncertain reliability). |
| Diffuse color over the section, including spaces outside identifiable cells. | A widespread deposit does not resolve the expected intracellular pattern (UniProt P45974: cytoplasm, nucleus; HPA: tissue IHC). Consider nonspecific detection, incomplete blocking or inadequate washing, and compare a no-primary control processed with the same detection reagents (general IHC practice). |
| No convincing signal in Purkinje cells or cells in seminiferous ducts. | Those are HPA's reported High staining examples, making them useful checks for a weak run (HPA: tissue IHC). Review tissue preservation, retrieval, antibody application and detection controls before interpreting the absence as biology (general IHC practice). HPA's Uncertain IHC rating limits how definitive any single comparison can be (HPA: IHC reliability). |
💡Expected USP5 appearanceCall a section positive when identifiable Purkinje cells or cells in seminiferous ducts show clear cytoplasmic and nuclear staining, consistent with HPA's High calls; isolated membrane or extracellular deposits are suspect against USP5's reported intracellular location (HPA: tissue IHC; UniProt P45974: subcellular location, topology).
| IHC evidence strength | HPA labels tissue IHC Uncertain because antibody staining and RNA expression have low consistency; external verification is pending (HPA: IHC reliability). Use its cell-level pattern as an expectation, then weigh morphology and controls before treating intensity differences as biological differences (general IHC practice). |
| Intracellular distribution | UniProt places USP5 in the cytoplasm, nucleus and stress granules, with no transmembrane segment (UniProt P45974: subcellular location, topology). HPA tissue IHC describes cytoplasmic and nuclear staining; its routine tissue profile does not establish a stress-granule pattern in a given section (HPA: tissue IHC). |
| Cell population and tissue context | HPA calls staining High in Purkinje cells and cells in seminiferous ducts, Medium in several listed glandular, hematopoietic, respiratory epithelial and neuronal populations, and Not detected in selected cell populations (HPA: tissue IHC). Score the identified population rather than assigning one intensity to an entire organ (general IHC practice). |
| Isoform coverage | UniProt lists 2 isoforms, Long and Short (UniProt P45974: isoforms). The supplied evidence gives no antibody epitope or isoform-specific IHC validation, so a staining difference cannot be attributed to one isoform from this record alone (UniProt P45974: isoforms; HPA: antibody validation). |
| IF/ICC Q&A: where should signal appear? | Nucleoplasm and cytosol are HPA's approved ICC-IF locations (HPA: subcellular). That supports an intracellular localisation check, while HPA rates the listed antibody's IHC evidence Uncertain and its ICC evidence Approved (HPA: antibody validation). ICC-IF image cell lines do not establish an IHC tissue intensity (HPA: subcellular; HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| The expected positive cell population is blank. | A missed IHC step, weak detection or unsuitable processing can suppress visible staining (general IHC practice); HPA reports High staining in Purkinje cells and cells in seminiferous ducts (HPA: tissue IHC). | Confirm the correct cell population on the section, then check the run's positive control, retrieval record, antibody application and detection sequence (general IHC practice). Avoid declaring that USP5 is absent from a specimen on the basis of one failed run (HPA: IHC reliability, Uncertain). |
| Nuclei alone or cytoplasm alone stain. | HPA describes both compartments in tissue IHC, although the apparent balance can vary with cell morphology and detection conditions (HPA: tissue IHC; general IHC practice). | Check that the apparent compartment is inside intact, identifiable cells and compare with a positive tissue area on the same run (general IHC practice). Record the observed distribution; do not force a dual-compartment score when one compartment is unclear (HPA: tissue IHC). |
| Membrane rims or extracellular deposits dominate. | The pattern conflicts with USP5's cytoplasmic and nuclear annotation and its lack of a transmembrane segment (UniProt P45974: subcellular location, topology). | Inspect section edges and deposits, compare the no-primary control, and review blocking, washing and detection reagents (general IHC practice). Reassess intracellular staining before calling the rim USP5-positive (UniProt P45974: subcellular location). |
| Cells reported as not detected show strong color. | HPA reports no detection in salivary glandular cells, smooth muscle cells and soft tissue fibroblasts; cross-reactivity or endogenous detection activity is possible (HPA: tissue IHC; general IHC practice). | Verify cell identity and compare no-primary and detection controls; investigate persistent cell-associated signal with an independent specificity check if available (general IHC practice). Treat the HPA comparison cautiously because its IHC reliability is Uncertain (HPA: IHC reliability). |
| Diffuse background obscures cell boundaries. | Nonspecific reagent binding, endogenous detection activity or inadequate washing can produce broad chromogenic background (general IHC practice). | Review blocking and washes, and compare a no-primary control under the same chromogenic detection conditions (general IHC practice). Score only signal that can be assigned to identifiable cytoplasm or nuclei (HPA: tissue IHC; general IHC practice). |
| IF/ICC localisation and tissue IHC impressions disagree. | HPA approves nucleoplasm and cytosol as ICC-IF locations, but rates tissue IHC Uncertain; the assays and specimens provide different kinds of evidence (HPA: subcellular; HPA: antibody validation). | Check each result against its own controls and cell morphology (general IHC/IF practice). Use the IF/ICC result to assess localisation, while retaining HPA's tissue-specific IHC intensity calls and their reliability caveat for the paraffin section (HPA: subcellular; HPA: tissue IHC). |
Experiment evidence
IHC & IF images showing USP5 staining
A04550-1 · IHCIHC analysis of USP5 using anti-USP5 antibody (A04550-1). USP5 was detected in a paraffin-embedded section of human breast cancer tissue. He
A04550-1 · IHCIHC analysis of USP5 using anti-USP5 antibody (A04550-1). USP5 was detected in a paraffin-embedded section of human testis tissue. Heat medi
A04550-1 · IFIF analysis of USP5 using anti-USP5 antibody (A04550-1). USP5 was detected in an immunocytochemical section of A549 cells. Enzyme antigen re
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000111667-USP5/tissue/cerebral+cortex#imid_2259756
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000111667-USP5/tissue/colon#imid_2259698
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000111667-USP5/tissue/liver#imid_2259683
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000111667-USP5/tissue/kidney#imid_2259692
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA006756; green = USP5, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA006756; green = USP5, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for USP5 IHC & IF
🧪Run cerebellum first and confirm staining in Purkinje cells (HPA: High in Purkinje cells); use salivary gland as the negative tissue (HPA: Not detected in salivary gland glandular cells). On the positive slide, neighboring cells without visible signal should remain at background, but their USP5 status should not be assumed from the supplied HPA row (HPA: High in Purkinje cells).
Positive control tissue: Cerebellum (Purkinje cells, HPA High)
Negative control tissue: Salivary gland (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show USP5 in A-431, U-251MG, U2OS, NIH 3T3, with annotated localisation: Nucleoplasm (approved), Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a host-species- and immunoglobulin-class-matched isotype control; compare these with USP5 knockout material processed in parallel as a biological negative (standard IHC practice). For chromogenic staining, quench endogenous peroxidase and check the cerebellum control for residual background before interpreting DAB signal (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A04550-1 paraffin-section caption does not report its fixative (selected-SKU tissue-IHC caption: fixative not stated). The caption uses heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required; it provides direct support for paraffin IHC, while the supplied ICC-IF images do not establish that frozen-section IF is easier (selected-SKU tissue-IHC caption: EDTA pH 8.0; HPA subcellular: ICC-IF images). If cerebellum is used for IF, check an unstained section for neuronal autofluorescence before interpreting signal (standard IF practice).
HPA tissue IHC evidence for USP5
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Low 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 |
|---|---|---|---|---|
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Testis | Cells in seminiferous ducts | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced USP5 IHC Tips
These questions address chromogenic USP5 staining in paraffin sections, using the A04550-1 tissue example and the stated limits of the localisation evidence.
How should I optimise retrieval if USP5 staining is weak in paraffin sections?
Start with heat-mediated retrieval in EDTA pH 8.0 for paraffin sections (datasheet A04550-1). Keep the documented 2 μg/mL primary antibody and overnight incubation at 4°C constant while testing retrieval (datasheet A04550-1). Compare adjacent sections heated for 10, 15, and 20 minutes, allowing consistent cooling before staining and checking tissue integrity (standard IHC practice). If staining remains weak, test citrate pH 6.0 as a fallback on adjacent sections, changing only the retrieval condition (standard IHC practice). Assess nuclear and cytoplasmic signal against a no-primary control before choosing a condition (UniProt P45974 subcellular; standard IHC practice).
Could fixation explain weak or uneven USP5 staining?
The A04550-1 tissue caption identifies a paraffin-embedded section but does not state its fixative, so target-specific fixation sensitivity is unknown (datasheet A04550-1). Record the fixative, fixation duration, tissue thickness, and processing history for each specimen before comparing staining intensity (standard IHC practice). On comparable material, examine whether weak staining tracks with prolonged fixation or with poorly preserved morphology, while holding retrieval and detection constant (standard IHC practice). Include an internally preserved region and a no-primary control to assess signal and background (standard IHC practice). Treat any fixation effect as an observed result of that comparison, not an established property of USP5 (datasheet A04550-1).
Should USP5 staining appear in nuclei, cytoplasm, or both?
Evaluate both nuclear and cytoplasmic staining: USP5 is annotated in the nucleus and cytoplasm, including stress granules (UniProt P45974 subcellular). Independent cell imaging places USP5 in the nucleoplasm and cytosol, supporting a two-compartment review of chromogenic sections (HPA subcellular). Score the compartments separately in intact cells, using a counterstain that leaves nuclear boundaries readable (standard IHC practice). A strictly membranous pattern warrants scrutiny because USP5 has no transmembrane segment, although that observation alone cannot establish specificity (UniProt P45974 topology; standard IHC practice). Compare any punctate cytoplasmic pattern with morphology and controls before attributing it to stress granules (UniProt P45974 subcellular; standard IHC practice).
How can isoforms or epitope location affect my IHC result?
USP5 has two annotated isoforms, Long and Short, but the supplied A04550-1 tissue caption does not map the antibody epitope (UniProt P45974 isoforms; datasheet A04550-1). Obtain the immunogen sequence and compare it with both isoforms before claiming that staining measures total USP5 (standard IHC practice). The annotated USP domain spans residues 326–856, with UBA regions at 654–695 and 722–762 (UniProt P45974 domains). Several modified residues are annotated, including positions 149, 156, 292, and 623, but their effect on this antibody is unknown (UniProt P45974 modified residues; datasheet A04550-1). Use an independent epitope antibody or a specificity control when an isoform-specific interpretation matters (standard IHC practice).
How would I assess USP5 by IF alongside a cell-type marker?
Treat IF as a separate optimisation: the supplied A04550-1 tissue example documents chromogenic IHC in a paraffin section, without an IF protocol (datasheet A04550-1). Multiplex USP5 with a validated Purkinje-cell marker when examining cerebellum, while recognising that the reported Purkinje-cell staining has uncertain reliability (HPA tissue IHC; standard IF practice). Choose a far-red fluorophore for a tissue with substantial short-wavelength autofluorescence, and inspect single-stain and no-primary controls (standard IF practice). Because USP5 is intracellular and lacks a transmembrane segment, test gentle permeabilisation to access cytosolic or nuclear epitopes, with antibody epitope accessibility confirmed empirically (UniProt P45974 topology and subcellular; standard IF practice). Keep nuclear and cytosolic measurements separate (HPA subcellular).
How can I distinguish USP5 staining from chromogenic background?
The documented A04550-1 example used 10% goat-serum blocking, a peroxidase-conjugated secondary antibody, and DAB development (datasheet A04550-1). Add a no-primary section and an endogenous-peroxidase blocking step, then compare signal with matched exposure and development times (standard IHC practice). If diffuse brown staining persists, titrate the documented 2 μg/mL primary concentration downward on adjacent sections and check secondary-only background (datasheet A04550-1; standard IHC practice). Examine tissue edges, damaged areas, and precipitate before assigning staining to cells (standard IHC practice). Retain a nuclear or cytoplasmic pattern only when it exceeds controls and follows intact-cell boundaries (UniProt P45974 subcellular; standard IHC practice).
What is a defensible way to quantify USP5 IHC? ⚠ ANSWER MARKED FOR VERIFICATION
Score nuclear and cytoplasmic USP5 separately because both compartments are supported by localisation evidence (UniProt P45974 subcellular; HPA subcellular). For each compartment, report the percentage of positive intact cells and an H-score calculated as 1 × % weak + 2 × % moderate + 3 × % strong, giving a 0–300 scale (standard IHC practice). Define thresholds using the same staining run and its negative controls, and apply them consistently across sections (standard IHC practice). Normalise cell counts to eligible cells or sampled tissue area, stating the denominator and excluding necrotic or damaged regions (standard IHC practice). Report cell type and region because the tissue staining profile varies and its reliability is uncertain (HPA tissue IHC).
When is an apparent USP5-positive region likely to be artefactual?
Give greatest weight to signal inside intact nuclear or cytoplasmic cell boundaries, the compartments supported for USP5 (UniProt P45974 subcellular; HPA subcellular). Suspect artefact when brown signal concentrates at section edges, in necrotic areas, or outside identifiable cells (standard IHC practice). A membranous-only pattern needs independent confirmation because USP5 has no annotated transmembrane segment (UniProt P45974 topology; standard IHC practice). Compare suspect regions with no-primary and peroxidase-block controls to identify background from detection chemistry (standard IHC practice). Use reported cell patterns as context, not proof of identity: HPA rates its tissue IHC evidence uncertain because staining and RNA expression have low consistency (HPA tissue IHC).
Boster reagents
Best USP5 / Ubiquitin carboxyl-terminal hydrolase 5 IHC Antibodies
The catalog includes USP5 antibodies for human paraffin-section IHC and A549-cell IF/ICC, with images for A04550-1 (A04550-1 image captions). Both list human, mouse, and rat reactivity (catalog).
Real IHC data
Anti-USP5 Antibody ®
Cat # A04550-1
A04550-1 has IHC images from human breast cancer and testis paraffin sections and an IF image from A549 cells (A04550-1 image captions). M04550-3 lists IF/ICC and human, mouse, and rat reactivity, but has no supplied IHC or IF image (catalog: M04550-3).
Which to pick: Choose A04550-1 for tissue IHC: its images document human paraffin sections with EDTA retrieval at pH 8.0 and primary antibody at 2 μg/ml; the fixative is unreported (A04550-1 IHC image captions). For IF/ICC, A04550-1 has an A549-cell IF image at 5 μg/ml, while rabbit monoclonal M04550-3 lists IF/ICC at 1:50–1:200 without a supplied image (A04550-1 IF image caption; catalog: M04550-3). Both list human, mouse, and rat reactivity, but the supplied tissue IHC images establish conditions only for human samples with A04550-1 (catalog; A04550-1 IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P45974 (UBP5_HUMAN, Ubiquitin carboxyl-terminal hydrolase 5).
- Human Protein Atlas. USP5 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. USP5 subcellular location (ICC-IF): Localized to the nucleoplasm and cytosol..
- Human Protein Atlas. USP5 antibody validation summary (1 antibodies).
- Ubiquitin specific peptidase 5 regulates colorectal cancer cell growth by stabilizing Tu translation elongation factor. Theranostics 2019 — PMC6592179.
- Deubiquitinase inhibitor degrasyn suppresses metastasis by targeting USP5-WT1-E-cadherin signalling pathway in pancreatic ductal adenocarcinoma. Journal of cellular and molecular medicine 2020 — PMC6991651.
- USP5 facilitates bladder cancer progression by stabilizing the c-Jun protein. Cancer cell international 2024 — PMC10792936.
- EOAI, a ubiquitin-specific peptidase 5 inhibitor, prevents non-small cell lung cancer progression by inducing DNA damage. BMC cancer 2023 — PMC9826599.
- PubMed PMID:7498549 — UniProt-cited evidence.
- PubMed PMID:8723724 — UniProt-cited evidence.
- PubMed PMID:9074930 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for USP5 (Ubiquitin carboxyl-terminal hydrolase 5). Expected localisation Cytoplasmic and nuclear staining in several tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A04550-1). Validated IHC antibody: A04550-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Cerebellum (Purkinje cells); negative Salivary gland. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A04550-1); 2. Fixation: Image fixative and duration unreported (datasheet A04550-1); 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 A04550-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A04550-1); 8. Primary antibody: Rabbit anti-USP5, 2-5 μg/ml (datasheet A04550-1); 9. Primary incubation: Overnight at 4 °C (datasheet A04550-1). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A04550-1); Caveat: Low staining–RNA concordance; validate the tissue pattern (HPA tissue IHC); Isoform / epitope: 2 isoforms (Long, Short); epitope coverage is unknown (UniProt); Regulation: No abundance regulator annotated (UniProt). Evidence: UniProt P45974, HPA tissue IHC · Uncertain, HPA ICC-IF · nucleoplasm and cytosol., Datasheet A04550-1, PMC6592179, PMC6991651. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/USP5/immunohistochemistry-ihc-guide.