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NEDD8 / Ubiquitin-like protein NEDD8 · IHC design guide
Design Immunohistochemistry for NEDD8
Plan chromogenic IHC for NEDD8 using the catalog antibody’s paraffin-section protocol (datasheet A00547). Compare nuclear and cytoplasmic staining against a high-staining tissue such as bronchial respiratory epithelium, while accounting for the uncertain reliability of the tissue profile (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 tissue staining (HPA tissue IHC) | |
| Staining pattern | Nuclear and cytoplasmic staining in most tissues (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). Selected-image fixative and duration unreported (datasheet A00547); verify before use. | |
| Caveat | Tissue staining has uncertain RNA concordance (HPA tissue IHC) | |
| Regulation | Broad tissue expression; no stimulus response reported (HPA tissue IHC) | |
| Isoform / epitope | No annotated isoforms; mature chain spans residues 1–76 (UniProt) |
Section 1
Recommended NEDD8 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is paired with three published NEDD8 IHC methods (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 noteFor paraffin sections, start with citrate pH 6 heat retrieval (A00547 datasheet). The published excerpts do not specify a retrieval method (PMC12960655; PMC6501157; PMC9830515).
Section 2
What Is the Expected NEDD8 Staining Pattern?
NEDD8 staining should be predominantly nuclear, with cytoplasmic staining possible in tissue sections (UniProt Q15843: mainly nuclear; HPA tissue IHC: cytoplasmic and nuclear expression in most tissues). Respiratory epithelial cells in bronchus and nasopharynx, Purkinje cells, cortical neurons, and parathyroid glandular cells show high staining (HPA tissue IHC). Interpret this pattern cautiously: HPA rates tissue IHC reliability Uncertain, pending external verification (HPA tissue IHC). NEDD8 has no transmembrane segment (UniProt Q15843 topology).
| Nuclear staining, with some cytoplasmic staining, in bronchial respiratory epithelium. | This fits a high-staining reference cell population (HPA tissue IHC: High in bronchial respiratory epithelial cells) and the reported compartment pattern (HPA tissue IHC: cytoplasmic and nuclear; UniProt Q15843: mainly nuclear). It is supportive, rather than definitive, because tissue IHC reliability is Uncertain (HPA tissue IHC). |
| Staining appears only at cell borders or outside cells, with no convincing nuclear signal. | An exclusively membranous or extracellular pattern conflicts with the mainly nuclear annotation and the reported tissue IHC pattern (UniProt Q15843: mainly nuclear, no transmembrane segment; HPA tissue IHC: cytoplasmic and nuclear). Review localization and controls before assigning it to NEDD8 (general IHC practice). |
| Strong staining occurs in alveolar cells while the expected respiratory epithelial cells are faint. | The cell distribution conflicts with the supplied reference: alveolar cells are Low, whereas bronchial and nasopharyngeal respiratory epithelial cells are High (HPA tissue IHC). Cross-reactivity or endogenous detection activity is possible; morphology and detection controls can help distinguish them (general IHC practice). |
| Uniform color covers nuclei, cytoplasm, and tissue spaces without clear cell boundaries. | This is more consistent with diffuse background than an interpretable cell pattern (general IHC practice). The HPA tissue profile describes cellular cytoplasmic and nuclear expression; it does not validate color deposited across tissue spaces (HPA tissue IHC). |
| No staining is visible in a bronchus section with identifiable respiratory epithelium. | That is unexpected for this reference population (HPA tissue IHC: High in bronchial respiratory epithelial cells). Check tissue preservation, detection controls, and assay conditions before calling the specimen NEDD8-negative (general IHC practice); the HPA IHC assessment itself remains Uncertain (HPA tissue IHC). |
💡Expected NEDD8 appearanceA plausible positive IHC result shows clear nuclear staining, potentially accompanied by cytoplasmic staining, in bronchial respiratory epithelial cells with relatively high signal (UniProt Q15843: mainly nuclear; HPA tissue IHC: High in bronchial respiratory epithelial cells; HPA tissue IHC: cytoplasmic and nuclear); isolated staining of tissue spaces or an exclusively cell-border pattern warrants investigation as possible background or off-target signal (general IHC practice; UniProt Q15843 topology).
| Choice of reference cells | HPA reports High staining in bronchial and nasopharyngeal respiratory epithelium, Purkinje cells, cortical neurons, and parathyroid glandular cells; alveolar cells, cholangiocytes, cardiomyocytes, smooth muscle cells, myocytes, and fibroblasts are Low (HPA tissue IHC). Choose and score the specified cells within each tissue (general IHC practice). |
| Transcript–stain comparison | UniProt lists heart and skeletal muscle among tissues with high NEDD8 expression, while HPA scores cardiomyocytes and skeletal myocytes Low by tissue IHC (UniProt Q15843 tissue specificity; HPA tissue IHC). These statements concern different readouts and do not justify upgrading those cells to high-staining IHC controls (general interpretation). |
| Subcellular interpretation | UniProt describes NEDD8 as mainly nuclear; HPA tissue IHC reports nuclear and cytoplasmic expression in most tissues (UniProt Q15843 subcellular location; HPA tissue IHC). Nuclear enrichment is therefore useful, but cytoplasmic staining alone cannot be dismissed without checking cell type and controls (general IHC practice). |
| NEDD8 processing and conjugation | The annotated precursor is 81 amino acids, with residues 77–81 removed and residues 1–76 forming mature NEDD8; NEDD8 can conjugate to substrates including cullins and p53 (UniProt Q15843 processing and function). These facts do not specify this antibody's epitope or predict its staining intensity (UniProt Q15843; antibody epitope not supplied). |
| Evidence and antibody validation | HPA rates tissue IHC reliability Uncertain, with medium antibody–RNA consistency pending external verification; CAB004082 has an Uncertain IHC status, while IHC status is not listed for HPA027583 or HPA060254 (HPA tissue IHC; HPA antibodies). Interpret apparent positives alongside controls (general IHC practice). |
| IF/ICC: should its pattern match IHC? | The separate ICC-IF evidence supports nucleoplasmic localization and labels additional Golgi and cytosolic localization Uncertain (HPA subcellular). Tissue IHC reports nuclear and cytoplasmic staining (HPA tissue IHC). Compare the compartment-level observations cautiously; these are different applications (general interpretation). |
| Situation | Likely cause | Next action |
|---|---|---|
| A reference section is entirely blank. | The absent signal conflicts with High staining reported for bronchial respiratory epithelial cells (HPA tissue IHC); the failure point cannot be identified from that observation alone (general IHC practice). | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. |
| Respiratory epithelium stains weakly, but nearby low-reference cells stain strongly. | This reverses the reported comparison between bronchial respiratory epithelial cells and alveolar cells, if those are the cells present (HPA tissue IHC: High versus Low). Cell identification or nonspecific detection may explain the mismatch (general IHC practice). | Recheck morphology and scoring areas, then compare with a no-primary detection control and a separate known-positive section (general IHC practice). Treat the HPA profile as a guide because its tissue IHC reliability is Uncertain (HPA tissue IHC). |
| Signal forms a broad haze across cells and tissue spaces. | Diffuse deposition can obscure cellular localization (general IHC practice); tissue spaces are not part of the reported nuclear and cytoplasmic cell pattern (HPA tissue IHC). | Review background on the no-primary control and assess blocking, antibody concentration, washes, and chromogen development using routine IHC controls (general IHC practice). Re-score only distinguishable cells (general IHC practice). |
| Color is confined to cell borders or extracellular material. | An exclusively border or extracellular pattern is inconsistent with mainly nuclear NEDD8 and its lack of a transmembrane segment (UniProt Q15843 subcellular location and topology). | Verify the compartment with a nuclear counterstain and cell morphology, and compare detection controls before accepting it as NEDD8 staining (general IHC practice). |
| Cardiomyocytes or skeletal myocytes show little staining. | Low staining in these cells agrees with the HPA tissue IHC entries, despite UniProt listing heart and skeletal muscle among tissues with high expression (HPA tissue IHC: Low in cardiomyocytes and myocytes; UniProt Q15843 tissue specificity). | Score the named cell populations against IHC reference patterns rather than treating tissue-level expression as an IHC intensity prediction (general interpretation; HPA tissue IHC). Use a High reference cell population for a stronger positive comparison (HPA tissue IHC). |
| The IHC slide differs from an ICC-IF image. | HPA supports nucleoplasmic ICC-IF localization but marks its additional Golgi and cytosolic locations Uncertain; tissue IHC reports cytoplasmic and nuclear staining (HPA subcellular; HPA tissue IHC). | Compare nuclear and cytoplasmic compartments within each application and inspect its own controls (general IHC/IF practice). Do not use an uncertain ICC-IF Golgi assignment as a required IHC pattern (HPA subcellular). |
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; respiratory epithelial cells should stain (HPA: High in bronchus respiratory epithelial cells). HPA detects NEDD8 in all 45 scored tissues, so use no-primary and isotype controls for the negative; no validated NEDD8-negative cell type is identified within the positive slide, and unstained cells, if present, should be judged against those controls (HPA: no negative tissue rows).
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: Use a no-primary (secondary-only) control, a concentration-matched rabbit IgG isotype control, and a biological specificity control such as NEDD8 knockout material or validated peptide competition (caption: rabbit primary; standard IHC controls). For the biotin-based DAB method, quench endogenous peroxidase and check for endogenous biotin background (caption: biotinylated secondary, SABC and DAB).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A00547 paraffin-section caption does not state the fixative (caption: fixative not stated). The caption uses heat retrieval in citrate buffer at pH 6 for 20 minutes, but does not establish that retrieval is required (caption: citrate retrieval). Whether frozen sections or IF are easier is unreported; in bronchus, distinguish epithelial staining from background due to endogenous peroxidase or biotin in the chromogenic method (HPA: High in bronchus respiratory epithelial cells; caption: SABC-DAB).
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
These answers use the A00547 paraffin-section IHC caption, UniProt Q15843 and HPA observations to guide chromogenic NEDD8 staining and interpretation.
What retrieval should I use if NEDD8 staining is weak in paraffin sections?
Start with heat-mediated citrate buffer at pH 6 for 20 minutes (A00547 tissue-IHC caption). The caption documents this retrieval for paraffin-embedded human lung cancer tissue, followed by overnight incubation at 4°C with 1 μg/mL antibody (A00547 tissue-IHC caption). If signal is weak, first check heating consistency, section adhesion and whether the positive control developed before changing retrieval conditions (standard IHC practice). If you then test another retrieval condition, compare it beside citrate pH 6 on matched sections and assess background as well as signal (standard IHC practice).
How should I troubleshoot fixation-related loss of NEDD8 staining?
The A00547 tissue-IHC caption identifies paraffin-embedded sections but does not state their fixative, so target-specific fixation sensitivity is unknown (A00547 tissue-IHC caption). Record the fixative, fixation duration and processing history for each block, then compare matched sections using the same citrate pH 6, 20-minute retrieval (A00547 tissue-IHC caption; standard IHC practice). Include a previously staining control section in the same run to distinguish a block-specific problem from a detection problem (standard IHC practice). Interpret any fixation effect only after that comparison; the reported staining pattern and NEDD8 sequence features do not establish fixation tolerance (HPA tissue IHC; UniProt Q15843; standard IHC practice).
Should I expect nuclear or cytoplasmic NEDD8 staining in IHC?
Assess nuclear and cytoplasmic staining separately: UniProt describes NEDD8 as mainly nuclear, while HPA tissue IHC reports both compartments in most tissues (UniProt Q15843 subcellular; HPA tissue IHC). HPA subcellular imaging supports nucleoplasmic localisation; its additional cytosolic and Golgi assignments are uncertain (HPA subcellular). NEDD8 has no annotated transmembrane segment, so a sharply membrane-restricted IHC pattern warrants a specificity check rather than a membrane assignment (UniProt Q15843 topology; standard IHC practice). Compare compartment patterns with a matched control section processed using citrate pH 6 retrieval for 20 minutes (A00547 tissue-IHC caption; standard IHC practice).
Could NEDD8 processing or conjugation change what this antibody detects?
NEDD8 is an 81-amino-acid precursor whose annotated propeptide spans residues 77–81; the mature chain spans 1–76 (UniProt Q15843 processing). UniProt lists 0 isoforms and describes covalent attachment to substrates including cullins and p53/TP53 (UniProt Q15843 isoforms and function). The A00547 tissue-IHC caption does not define the antibody epitope or establish whether it distinguishes free from conjugated NEDD8 (A00547 tissue-IHC caption). Therefore, score staining as NEDD8 immunoreactivity and use a separate specificity experiment before assigning a particular molecular form; keep citrate pH 6 retrieval constant during that comparison (A00547 tissue-IHC caption; standard IHC practice).
How can I investigate NEDD8 localisation by IF alongside this IHC assay?
Treat IF as a separate assay: the supplied A00547 evidence describes paraffin-section chromogenic IHC, not an IF staining condition (A00547 tissue-IHC caption). For multiplex IF, pair NEDD8 with a marker of the cell type under study and compare nuclear signal with a DNA counterstain; NEDD8 is mainly nuclear by UniProt annotation (UniProt Q15843 subcellular; standard IF practice). Choose fluorophores after checking tissue autofluorescence and include single-label controls to assess bleed-through (standard IF practice). For intracellular nuclear and cytosolic epitopes, optimise permeabilisation after fixation; NEDD8 has no annotated transmembrane segment, and the IHC caption supplies no IF fixation condition (UniProt Q15843 topology; A00547 tissue-IHC caption; standard IF practice).
How do I reduce diffuse brown background without masking NEDD8?
First inspect a no-primary control and confirm that the same tissue regions develop colour without the catalog antibody (standard IHC practice). The documented workflow used 10% goat serum block, 1 μg/mL rabbit antibody overnight at 4°C, and biotinylated secondary followed by SABC and DAB (A00547 tissue-IHC caption). Check washing, secondary binding and the avidin–biotin detection steps; apply a peroxidase block when using DAB as a general chromogenic IHC measure (standard IHC practice). Judge any change against tissue morphology and compartment pattern, since HPA reports both nuclear and cytoplasmic NEDD8 staining in most tissues (HPA tissue IHC; standard IHC practice).
What should I score when comparing NEDD8 staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue region and cell population before scoring, and exclude folds, necrosis and section edges from analysis (standard IHC practice). Record nuclear and cytoplasmic results separately using percentage of positive cells plus intensity, or an H-score from 0–300 for each compartment (standard IHC scoring; HPA tissue IHC). Normalise positive-cell counts to the number of evaluable cells in the defined region, or report positive-cell density per mm² of evaluable tissue (standard IHC practice). Keep retrieval at citrate pH 6 for 20 minutes and detection settings consistent across compared sections; HPA rates its tissue staining reliability as uncertain (A00547 tissue-IHC caption; HPA tissue IHC; standard IHC practice).
How can I distinguish credible NEDD8 staining from an artefact?
A credible result should follow cell boundaries and show a reproducible compartment pattern, with nuclear signal plausible because NEDD8 is mainly nuclear (UniProt Q15843 subcellular; standard IHC practice). Cytoplasmic staining can also be plausible, but HPA rates its tissue-IHC reliability uncertain and reports both nuclear and cytoplasmic expression in most tissues (HPA tissue IHC). Treat staining confined to cut edges or necrotic areas, or reproduced in a no-primary control, as suspect; investigate endogenous peroxidase signal in the DAB workflow (standard IHC practice; A00547 tissue-IHC caption). Compare a same-run control after citrate pH 6, 20-minute retrieval before attributing differences to NEDD8 biology (A00547 tissue-IHC caption; standard IHC practice).
Boster reagents
Best NEDD8 / Ubiquitin-like protein NEDD8 IHC Antibodies
A00547 has IHC data from a human paraffin tissue section 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 will render with an IHC figure from a paraffin section of human lung cancer tissue; its separate IF/ICC caption documents A431 cells (A00547 image captions). M00547 lists IHC and IF/ICC applications and human, mouse and rat reactivity, but has no supplied IHC or IF figure (M00547 catalog applications, reactivity and image fields).
Which to pick: Choose A00547 for tissue IHC: its own caption documents citrate retrieval at pH 6 for 20 minutes and 1 μg/ml primary antibody overnight at 4°C in a human paraffin section; the fixative is unreported (A00547 IHC image caption). For IF/ICC, A00547 has an A431-cell figure using 2 μg/ml, while M00547 lists IF/ICC without a supplied figure (A00547 IF image caption; M00547 catalog applications and image fields). For mouse or rat samples, both list reactivity, and M00547 is rabbit monoclonal while A00547 has no clone specified; the supplied tissue IHC figure documents human tissue only (catalog reactivity, host and clone; A00547 IHC image caption).
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 tissue staining (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). Selected-image fixative and duration unreported (datasheet A00547); verify before use.; Caveat: Tissue staining has uncertain RNA concordance (HPA tissue IHC); Isoform / epitope: No annotated isoforms; mature chain spans residues 1–76 (UniProt); Regulation: Broad tissue expression; no stimulus response reported (HPA tissue IHC). 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.