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NEDD8 / Ubiquitin-like protein NEDD8 · IHC design guide
Design Immunohistochemistry for NEDD8
Plan NEDD8 IHC on paraffin sections using the reported nuclear and cytoplasmic tissue pattern, with respiratory epithelium as a high staining reference (HPA tissue IHC). Compare sections with consistent fixation and interpret intensity cautiously because tissue staining reliability is uncertain (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NEDD8 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 | Nuclear and cytoplasmic staining in most tissues (HPA tissue IHC) | |
| Staining pattern | Most tissues show nuclear and cytoplasmic signal (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A00547) | |
| Positive control | Bronchus+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Tissue staining has uncertain reliability (HPA tissue IHC) | |
| Regulation | Expression regulation is not established (UniProt) | |
| Isoform / epitope | No isoforms annotated; residues 77–81 are removed during maturation (UniProt) |
Section 1
Recommended NEDD8 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet: A00547) with published NEDD8 chromogenic IHC on frozen synovium, tissue sections, and paraffin breast arrays (PMC12960655; PMC6501157; PMC9830515).
| Sample | Paraffin-embedded human lung cancer tissues; fixative not specified (datasheet A00547) |
| Fixation | Image fixative and duration unreported (datasheet A00547); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet A00547) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A00547) |
| Primary antibody | Rabbit anti-NEDD8, 0.5-1μg/ml (datasheet A00547) |
| Primary incubation | Overnight at 4 °C (datasheet A00547) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A00547) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | NEDD8-positive staining in respiratory epithelial cells of bronchus (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and nuclear expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated citrate pH 6 retrieval for paraffin sections (datasheet: A00547). The frozen-section protocol instead specifies acetone fixation (PMC12960655).
Section 2
What Is the Expected NEDD8 Staining Pattern?
NEDD8 is mainly nuclear (UniProt Q15843: subcellular location) and has no transmembrane segment (UniProt Q15843: topology). In paraffin sections, expect nuclear and cytoplasmic staining in many tissues, with strong signal in selected epithelial, neuronal and glandular cells (HPA: tissue IHC). Treat this as a reference pattern, not a definitive specificity test: HPA rates the tissue IHC profile Uncertain because staining and RNA show only medium consistency (HPA: tissue IHC reliability).
| Nuclear staining, with some cytoplasmic staining, in respiratory epithelium. | This fits the reported tissue pattern (HPA: cytoplasmic and nuclear expression in most tissues). Respiratory epithelial cells stain High in bronchus and nasopharynx (HPA: tissue IHC). Judge intensity in the named cells, since an entire section need not stain uniformly (standard IHC interpretation). |
| Signal appears confined to an unexpected compartment across the section. | A pattern lacking the expected nuclear component warrants review because NEDD8 is mainly nuclear (UniProt Q15843: subcellular location). Check morphology, counterstain and detection controls before calling it artefact (standard IHC practice); cytoplasmic signal alone is not inherently false because HPA reports it in tissue IHC (HPA: tissue IHC profile). |
| The strongest signal is in cells other than the expected positive population. | For example, strong alveolar staining would conflict with the reported Low level in alveolar cells, while bronchial respiratory epithelium is High (HPA: tissue IHC). Review cell identity and controls; antibody cross-reactivity or endogenous detection activity are possible explanations, not diagnoses from one slide (standard IHC practice). |
| Brown deposit spreads across cells, stroma or the section edge. | Diffuse signal without a coherent cellular pattern cannot establish NEDD8 localization (standard IHC interpretation). Compare a control processed without primary antibody and inspect reagent and wash conditions for nonspecific staining or endogenous detection activity (standard IHC practice). |
| Little or no signal appears in a selected positive tissue. | Check that the expected cells are present: Purkinje cells in cerebellum and glandular cells in parathyroid are reported High (HPA: tissue IHC). A blank result may reflect the assay or sampling, and one blank section cannot overturn an HPA pattern rated Uncertain (HPA: tissue IHC reliability; standard IHC interpretation). |
💡Expected NEDD8 appearanceCall a section positive when identifiable cells show predominantly nuclear signal with permissible cytoplasmic staining, especially the High respiratory epithelial, Purkinje, neuronal or parathyroid glandular populations (UniProt Q15843: subcellular location; HPA: tissue IHC); diffuse deposit without cell boundaries is suspect (standard IHC interpretation).
| Choice of positive and lower-signal cells | Bronchus and nasopharynx respiratory epithelium, cerebellar Purkinje cells, cerebral cortical neurons and parathyroid glandular cells are High (HPA: tissue IHC). Alveolar cells, cholangiocytes, cardiomyocytes, myocytes and fibroblasts are Low comparators, not established negatives (HPA: tissue IHC). |
| Cell type within the same tissue | Interpret named cells rather than assigning one score to an organ: bronchial respiratory epithelial cells are High, whereas lung alveolar cells are Low (HPA: tissue IHC). A difference between those compartments can therefore be expected without treating either tissue as an absolute positive or negative control (HPA: tissue IHC). |
| NEDD8 form and localization | UniProt annotates an 81-aa precursor, removal of residues 77–81, and a mature 1–76 chain that conjugates to cellular proteins (UniProt Q15843: processing and function). The payload gives no antibody epitope, so it cannot establish which NEDD8 forms the IHC stain detects (UniProt Q15843: processing; supplied antibody data). |
| Strength of antibody evidence | The HPA tissue profile is Uncertain, and CAB004082 has Uncertain IHC validation; the other listed antibodies have no IHC status in the supplied record (HPA: tissue IHC reliability; HPA: antibody validation). Use morphology and controls when interpreting a surprising pattern (standard IHC practice). |
| IF/ICC Q: Where should fluorescence appear? | A: Mainly in the nucleoplasm (Supported); Golgi apparatus and cytosol are additional Uncertain locations (HPA: subcellular ICC-IF). This cellular imaging evidence helps interpret localization, while tissue intensity calls come from the separate HPA tissue IHC profile (HPA: subcellular ICC-IF; HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| No stain in a selected positive section | The named positive cells may be absent from the sampled area, or the staining run may have failed (HPA: tissue IHC; standard IHC practice). | Confirm the relevant cells on the counterstained section, then review the run's positive control, primary antibody, retrieval and detection steps (standard IHC practice). |
| Widespread diffuse brown background | Nonspecific reagent binding or endogenous detection activity can produce deposit that does not follow cell anatomy (standard IHC practice). | Inspect a control without primary antibody; review blocking, washes and the detection system before scoring NEDD8 (standard IHC practice). |
| Predominantly cytoplasmic staining with weak nuclei | Cytoplasmic tissue staining is reported, but a consistently absent nuclear component differs from NEDD8's main localization (HPA: tissue IHC profile; UniProt Q15843: subcellular location). | Check nuclear morphology and counterstain, then compare another section and appropriate controls; avoid assigning specificity from compartment alone (standard IHC practice). |
| Strong staining in a reported lower-signal cell population | Cell misidentification, cross-reactivity or endogenous detection activity may explain a mismatch with an HPA Low call (HPA: tissue IHC; standard IHC practice). | Verify cell identity and compare nearby expected positive cells plus a control without primary antibody; report the mismatch if it persists (standard IHC practice). |
| Weak signal in heart or skeletal muscle despite UniProt expression | UniProt reports high tissue expression, while HPA calls cardiomyocyte and skeletal myocyte IHC staining Low (UniProt Q15843: tissue specificity; HPA: tissue IHC). | Use the cell-level HPA calls for slide interpretation; check a reported High IHC cell population before judging the run (HPA: tissue IHC; standard IHC practice). |
| Different fields or sections yield conflicting scores | The field may contain different cell populations, and the HPA tissue IHC profile has Uncertain reliability (HPA: tissue IHC reliability; standard IHC interpretation). | Score identified cell types in comparable fields, document the compartment and intensity, and keep the uncertainty attached to conclusions (standard IHC practice; HPA: tissue IHC reliability). |
Experiment evidence
IHC & IF images showing NEDD8 staining
A00547 · IHCIHC analysis of NEDD8 using anti-NEDD8 antibody (A00547). NEDD8 was detected in paraffin-embedded section of human lung cancer tissues. Heat
A00547 · IFIF analysis of NEDD8 using anti-NEDD8 antibody (A00547). NEDD8 was detected in immunocytochemical section of A431 cells. Enzyme antigen retr
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000129559-NEDD8/tissue/cerebral+cortex#imid_1438916
HPA · Parathyroid glandHuman Protein Atlas IHC, Parathyroid gland. Source: https://www.proteinatlas.org/ENSG00000129559-NEDD8/tissue/parathyroid+gland#imid_1438807
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000129559-NEDD8/tissue/colon#imid_1438850
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000129559-NEDD8/tissue/liver#imid_1438835
HPA · ICC-IF Hep-G2Human Protein Atlas immunofluorescence, Hep-G2, antibody HPA027583; green = NEDD8, blue = nucleus, red = microtubules.
HPA · ICC-IF Hep-G2Human Protein Atlas immunofluorescence, Hep-G2, antibody HPA027583; green = NEDD8, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NEDD8 IHC & IF
🧪Run bronchus first and assess respiratory epithelial cells, which HPA scores High for NEDD8 (HPA: bronchus, respiratory epithelial cells, High). HPA detects NEDD8 in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and treat cells with little visible signal on the positive slide as an internal background reference, not proven NEDD8-negative cells (HPA: no negative rows; standard IHC practice).
Positive control tissue: Bronchus (Respiratory epithelial cells, HPA High)
Negative control tissue: None in HPA: NEDD8 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show NEDD8 in Hep-G2, MCF-7, U2OS, A-431, U-251MG, A-549, KOLF2.1J, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Run a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species and clonality where known; assess specificity with a NEDD8 knockout specimen or peptide block when a validated blocking peptide is available (standard IHC practice). For chromogenic detection, check endogenous peroxidase and, with the caption’s biotin-based detection, endogenous biotin background in bronchus (selected A00547 tissue-IHC caption; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected paraffin-section caption does not state a fixative (selected A00547 tissue-IHC caption). The caption uses citrate retrieval at pH 6 for 20 minutes, but does not establish that retrieval is required; frozen sections or IF cannot be judged easier from the supplied evidence (selected A00547 tissue-IHC caption). Bronchial epithelium is an HPA-supported positive compartment, while endogenous peroxidase or biotin can complicate chromogenic interpretation and tissue autofluorescence can complicate IF (HPA: bronchus, respiratory epithelial cells, High; standard IHC/IF practice).
HPA tissue IHC evidence for NEDD8
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — 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 |
|---|---|---|---|---|
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Nasopharynx | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Parathyroid gland | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: NEDD8 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced NEDD8 IHC Tips
Troubleshoot NEDD8 staining in paraffin sections by checking retrieval, compartment, cell type and detection controls before interpreting signal.
What should I change if NEDD8 staining is weak after antigen retrieval?
Start with heat-mediated citrate retrieval at pH 6 for 20 minutes on paraffin sections (datasheet A00547; A00547 tissue-IHC caption). The documented example used 1 μg/mL primary antibody overnight at 4°C, so check those conditions before changing retrieval (A00547 tissue-IHC caption). Keep section thickness, heating and cooling consistent across test slides, then compare staining with a matched no-primary control (standard IHC practice). If signal remains weak, test a modest change in heating time on adjacent sections while monitoring tissue damage (standard IHC practice). Score nuclear and cytoplasmic staining separately because both appear in tissue IHC (HPA tissue IHC profile).
Could fixation explain weak or uneven NEDD8 IHC staining?
Target-specific fixation sensitivity is unknown: the paraffin-section caption does not state its fixative, and the supplied record gives no fixation comparison (A00547 tissue-IHC caption; UniProt Q15843 record). Document the fixative and processing history for each specimen, and compare sections processed alike before attributing a difference to NEDD8 biology (standard IHC practice). Use citrate retrieval at pH 6 for 20 minutes as the documented starting condition (A00547 tissue-IHC caption). Inspect morphology and compare edge with center staining to identify uneven processing (standard IHC practice). The caption’s overnight 4°C incubation is evidence for incubation conditions, not fixation tolerance (A00547 tissue-IHC caption).
Should NEDD8 staining be nuclear, cytoplasmic, or both?
Evaluate nuclear staining first because NEDD8 is mainly nuclear in the supplied protein record (UniProt Q15843 subcellular annotation). Tissue IHC also reports cytoplasmic and nuclear expression in most tissues, although its staining reliability is uncertain (HPA tissue IHC profile and reliability). Record the fraction and intensity of each compartment separately rather than treating any brown signal as equivalent (standard IHC practice). Compare staining with nearby cells of known morphology and a no-primary control under the same detection conditions (standard IHC practice). Predominantly extracellular deposits, isolated section-edge staining or signal confined to damaged areas warrants a technical review before interpretation (standard IHC practice).
Can this stain distinguish processed NEDD8 from conjugated NEDD8?
Do not assign a brown signal to free versus protein-conjugated NEDD8 without epitope validation: NEDD8 is processed from an 81-amino-acid precursor to a 1–76 mature chain and conjugates to substrates (UniProt Q15843 processing and function). The supplied record lists 0 isoforms, but it does not establish what molecular forms this antibody recognizes in sections (UniProt Q15843 isoforms; A00547 tissue-IHC caption). Ask whether the antibody epitope lies in the mature chain or the removed 77–81 propeptide before interpreting loss of staining (UniProt Q15843 processing; standard antibody-validation practice). Report compartment and cell-specific staining, using matched controls to assess specificity (standard IHC practice).
How can IF help assess an ambiguous NEDD8 IHC pattern?
On a matched section, multiplex NEDD8 with a marker for the expected cell type, such as respiratory epithelial cells in bronchus, which show high tissue-IHC staining (HPA tissue IHC: bronchus respiratory epithelial cells). Choose a spectrally separated, preferably far-red fluorophore and inspect an unstained section for tissue autofluorescence before assigning colocalisation (standard IF practice). Because NEDD8 has no transmembrane segment and is mainly nuclear, permeabilisation should support access to intracellular and nuclear epitopes; titrate it against preserved morphology (UniProt Q15843 topology and subcellular annotation; standard IF practice). Nucleoplasmic localisation is supported by subcellular IF evidence, while reported cytosolic and Golgi localisation is uncertain (HPA subcellular).
How do I reduce diffuse DAB background without losing NEDD8 signal?
First compare a no-primary section with the stained section to separate detection-system background from primary-antibody-associated signal (standard IHC practice). The documented method used 10% goat serum, a biotinylated secondary antibody, a streptavidin–biotin complex and DAB (A00547 tissue-IHC caption). Include a peroxidase block and assess endogenous biotin where relevant to that detection chemistry, then verify that washes remove residual reagent (standard chromogenic IHC practice). Titrate primary antibody around the documented 1 μg/mL condition while retaining its overnight 4°C incubation for comparison (A00547 tissue-IHC caption; standard IHC practice). Check whether apparent background follows tissue edges, folds or damaged regions (standard IHC practice).
How should I quantify NEDD8 staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, since tissue IHC reports both nuclear and cytoplasmic NEDD8 staining (HPA tissue IHC profile). For each compartment, record percent positive cells and intensity, then calculate an H-score as the sum of percent cells at each intensity multiplied by its intensity grade (standard IHC scoring practice). Alternatively, report positive-cell density per mm² within a consistently defined tissue area (standard IHC quantification practice). Normalize to the number of evaluable cells or analyzed tissue area, excluding folds and necrotic regions by the same rule for every section (standard IHC practice). Keep imaging and DAB thresholds fixed across comparisons (standard IHC practice).
What makes a NEDD8-positive IHC result convincing?
A convincing pattern should follow intact cell morphology, with nuclear staining expected and cytoplasmic staining also reported in tissues (UniProt Q15843 subcellular annotation; HPA tissue IHC profile). Check whether the positive cells fit the sampled tissue: bronchial respiratory epithelial cells have high reported staining, whereas lung alveolar cells have low reported staining (HPA tissue IHC: bronchus respiratory epithelial cells and lung alveolar cells). Exclude section-edge concentration, necrotic deposits and signal retained in a no-primary control before calling a cell positive (standard IHC practice). Peroxidase or biotin-related background needs particular attention with the documented streptavidin–biotin and DAB detection method (A00547 tissue-IHC caption; standard IHC practice). Interpret differences cautiously because HPA tissue-IHC reliability is uncertain (HPA reliability).
Boster reagents
Best NEDD8 / Ubiquitin-like protein NEDD8 IHC Antibodies
A00547 has IHC data from human lung cancer paraffin sections and IF/ICC data from A431 cells (A00547 image captions). Both antibodies list Human, Mouse, and Rat reactivity (catalog reactivity).
Real IHC data
Anti-NEDD8 Antibody ®
Cat # A00547
A00547 lists IHC and IF/ICC, with images from human lung cancer paraffin sections and A431 cells, respectively (catalog applications; A00547 image captions). M00547 lists IHC and IF/ICC and Human, Mouse, and Rat reactivity, but has no supplied IHC or IF image (catalog applications, reactivity, and image alts).
Which to pick: Choose A00547 for tissue IHC: its caption documents human lung cancer paraffin sections, citrate pH 6 retrieval, and 1 μg/ml antibody; the fixative is unreported (A00547 IHC caption). For IF/ICC, A00547 has A431 cell data at 2 μg/ml, while M00547 lists IF/ICC without a supplied image (A00547 IF caption; M00547 catalog applications and image alts). Both list Mouse and Rat reactivity, although the supplied images do not show those species; M00547 is the rabbit monoclonal option, clone AOAE-14 (catalog reactivity, host, clone, and image alts).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q15843 (NEDD8_HUMAN, Ubiquitin-like protein NEDD8).
- Human Protein Atlas. NEDD8 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. NEDD8 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the Golgi apparatus and cytosol..
- Human Protein Atlas. NEDD8 antibody validation summary (3 antibodies).
- Altered fibroblast-like synoviocyte epigenetics is responsible for deficient NUB1 expression in rheumatoid arthritis. Scientific reports 2026 — PMC12960655.
- Neddylation of NFATc1 and Runx2 regulates osteoclast-osteoblast balance and represents a dual-action therapeutic target for postmenopausal osteoporosis. Experimental & molecular medicine 2026 — PMC13538646.
- MLN4924 Exerts a Neuroprotective Effect against Oxidative Stress via Sirt1 in Spinal Cord Ischemia-Reperfusion Injury. Oxidative medicine and cellular longevity 2019 — PMC6501157.
- Neddylation of HER2 Inhibits its Protein Degradation and promotes Breast Cancer Progression. International journal of biological sciences 2023 — PMC9830515.
- PubMed PMID:9694792 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:10318914 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NEDD8 (Ubiquitin-like protein NEDD8). Expected localisation Nuclear and cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A00547). Validated IHC antibody: A00547 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Bronchus (Respiratory epithelial cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung cancer tissues; fixative not specified (datasheet A00547); 2. Fixation: Image fixative and duration unreported (datasheet A00547); 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: Citrate pH 6 , 20 min (datasheet A00547); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A00547); 8. Primary antibody: Rabbit anti-NEDD8, 0.5-1μg/ml (datasheet A00547); 9. Primary incubation: Overnight at 4 °C (datasheet A00547). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Tissue staining has uncertain reliability (HPA tissue IHC); Isoform / epitope: No isoforms annotated; residues 77–81 are removed during maturation (UniProt); Regulation: Expression regulation is not established (UniProt). Evidence: UniProt Q15843, HPA tissue IHC · Uncertain, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A00547, PMC12960655, PMC6501157. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NEDD8/immunohistochemistry-ihc-guide.