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NUCB2 / Nucleobindin-2 · IHC design guide
Design Immunohistochemistry for NUCB2
Plan chromogenic NUCB2 IHC in paraffin sections using the cytoplasmic tissue pattern as a reference (HPA tissue IHC). Colon glandular cells show high staining, while kidney glomerular cells show no detected staining; a secreted variant can complicate comparison with RNA expression (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NUCB2 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 staining in most tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in glandular and ciliated cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Bone marrow+4 more · see all | |
| Negative control | Kidney+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | A secreted variant can weaken RNA–protein concordance (HPA tissue IHC) | |
| Regulation | Expression regulation is not specified (UniProt) | |
| Isoform / epitope | 2 isoforms; nesfatin-1 cleavage affects epitope choice (UniProt) |
Section 1
Recommended NUCB2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is followed by four published NUCB2 IHC protocols (PMC3852884; PMC6169241; PMC6388962; PMC5471050).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A02400); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-NUCB2, 1:100–1:500 starting range (standard) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | NUCB2-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 HIER at 95–98 °C for 20 min (page antigen-retrieval specification); the thyroid study used EDTA pH 8.0 (PMC6388962).
Section 2
What Is the Expected NUCB2 Staining Pattern?
NUCB2 staining is predominantly cytoplasmic across tissues, with strong IHC signal reported in selected glandular, ciliated and hematopoietic cells (HPA tissue IHC). A perinuclear Golgi accent is plausible because ICC-IF localizes NUCB2 to the Golgi (HPA subcellular) and UniProt lists Golgi, ER, cytoplasmic and secreted locations (UniProt P80303). Interpret intensity by cell type: HPA rates tissue IHC reliability Enhanced but notes medium agreement with RNA because a variant is secreted (HPA tissue IHC). NUCB2 has no transmembrane segment (UniProt P80303 topology).
| Strong cytoplasmic staining in colon glandular cells, with some perinuclear accent. | This fits a reported High cell type (HPA: High in colon glandular cells). A Golgi accent is plausible (HPA: Golgi in ICC-IF); its absence alone does not invalidate IHC. |
| Signal confined to nuclear chromatin, with little cytoplasmic staining. | Treat this as discordant with the predominant tissue IHC pattern (HPA: cytoplasmic in most tissues). UniProt lists the nuclear envelope, so distinguish an envelope rim from chromatin staining (UniProt P80303). |
| Signal in kidney glomerular cells despite little staining in adjacent tissue. | Glomerular cells are Not detected in the HPA tissue profile (HPA: kidney glomeruli). Check cell identification and no-primary control; persistent staining may reflect cross-reactivity or endogenous detection activity (general IHC practice). |
| Diffuse brown deposit across cells, stroma and blank slide areas. | An indiscriminate deposit cannot establish the reported cell-specific cytoplasmic pattern (HPA tissue IHC). Consider nonspecific reagent binding, endogenous enzyme activity or incomplete washing (general chromogenic IHC practice). |
| No signal in a section containing identifiable colon glandular cells. | Colon glandular cells are reported High (HPA: colon). First confirm that the cells are present, then check antibody and detection controls; one negative run does not establish absent NUCB2. |
💡Expected NUCB2 appearanceA convincing positive shows strong cytoplasmic staining in a reported High cell type such as colon glandular cells (HPA tissue IHC), possibly with a Golgi accent (HPA subcellular); chromatin-only or indiscriminate slide-wide deposit is suspect (HPA tissue IHC; general IHC practice).
| Choice of comparison cells | Use the named cell populations, not whole-organ averages: colon glandular cells are High, while kidney glomerular cells are Not detected (HPA tissue IHC). Those labels do not classify every cell in either organ. |
| Subcellular reference | Tissue IHC is described as predominantly cytoplasmic (HPA tissue IHC). ICC-IF has an enhanced Golgi location (HPA subcellular), while UniProt also lists ER, membrane, nuclear envelope and secretion (UniProt P80303); do not require an identical appearance in every assay. |
| Processing and secretion | UniProt annotates a signal peptide at residues 1–24, NUCB2 chain at 25–420 and nesfatin-1 chain at 25–106 (UniProt P80303). Epitope position could affect which form an antibody detects; the supplied sources do not identify the catalog antibody's epitope. |
| Isoforms and interpretation | Two isoforms are listed (UniProt P80303). The supplied evidence does not assign tissue IHC staining to an isoform, so avoid explaining a weak or absent section by isoform expression without separate evidence. |
| IHC preparation and detection | Antigen retrieval, blocking, antibody dilution and chromogen development are general IHC optimization variables (general IHC practice). No NUCB2-specific retrieval condition, dilution or fixation sensitivity is supplied here; use the validated assay instructions and controls. |
| IF/ICC Q&A: what pattern is expected? | Golgi localization is the main ICC-IF finding (HPA subcellular: Golgi, enhanced). The supplied tissue IHC profile supports cytoplasmic staining (HPA tissue IHC); compare each result within its own assay and cell context. |
| Situation | Likely cause | Next action |
|---|---|---|
| Reported High cells are unstained in an IHC run. | Possible assay failure or absent target cells; HPA reports High colon glandular staining (HPA: colon). | Confirm glandular cells on the counterstained section, then review the IHC-validated antibody instructions, reagent controls and chromogen development (general IHC practice). |
| Chromatin-only staining dominates. | It conflicts with predominantly cytoplasmic tissue IHC (HPA tissue IHC), although a nuclear-envelope location is annotated (UniProt P80303). | Inspect the staining boundary at higher magnification and compare a reported High cell type; run a no-primary control to assess detection background (general IHC practice). |
| Kidney glomerular cells appear strongly positive. | HPA reports glomerular cells Not detected (HPA: kidney), so cell misidentification, cross-reactivity or detection background deserves investigation. | Verify glomerular identity and compare a no-primary section; assess whether signal follows cells or persists as diffuse deposit (general IHC practice). |
| Diffuse deposit obscures cellular boundaries. | Nonspecific binding, endogenous enzyme activity or excessive chromogen development can produce widespread staining (general chromogenic IHC practice). | Inspect a no-primary control; review blocking, washing and development time against the validated assay instructions (general IHC practice). |
| Only perinuclear puncta stain, while broad cytoplasm is faint. | Golgi enrichment is consistent with ICC-IF (HPA subcellular), but tissue IHC is summarized as cytoplasmic (HPA tissue IHC). | Score the identified cell population and compare a reported High control; describe the restricted pattern rather than assuming it proves loss of NUCB2 elsewhere. |
| IHC and IF images seem to disagree. | HPA summarizes tissue IHC as cytoplasmic but ICC-IF as Golgi-localized (HPA tissue IHC; HPA subcellular); assay context and image resolution differ (general microscopy practice). | Compare localization within each assay and its controls; avoid assigning a processing form or isoform without epitope evidence (UniProt P80303 processing and isoforms). |
Experiment evidence
IHC & IF images showing NUCB2 staining
A02400 · IHCImmunohistochemistry of Nucleobindin-2 in rat brain tissue with Nucleobindin-2 antibody at 2.5 μg/ml.
A02400 · IFImmunofluorescence of Nucleobindin-2 in rat brain tissue with Nucleobindin-2 antibody at 20 μg/ml.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000070081-NUCB2/tissue/cerebral+cortex#imid_2637764
HPA · Salivary glandHuman Protein Atlas IHC, Salivary gland. Source: https://www.proteinatlas.org/ENSG00000070081-NUCB2/tissue/salivary+gland#imid_3915040
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000070081-NUCB2/tissue/colon#imid_3915026
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000070081-NUCB2/tissue/liver#imid_3915019
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB015931; green = NUCB2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB015931; green = NUCB2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NUCB2 IHC & IF
🧪Run bone marrow first: hematopoietic cells should stain (HPA: High in bone marrow hematopoietic cells). Use skeletal muscle myocytes as the negative tissue (HPA: Not detected in skeletal muscle myocytes); within the marrow section, cells without specific staining should show only the counterstain, although HPA does not identify a particular negative marrow cell type.
Positive control tissue: Bone marrow (Hematopoietic cells, HPA High)
Negative control tissue: Kidney (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show NUCB2 in A-431, U-251MG, U2OS, with annotated localisation: Golgi apparatus (enhanced) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and host- and clonality-matched isotype controls, plus NUCB2 knockout tissue as a biological negative where available (standard IHC practice). In bone marrow, block endogenous peroxidase before chromogenic detection because hematopoietic cells can contain peroxidase (standard IHC practice).
⚠️Feasibility: A NUCB2-specific fixation window or fixation effect is unreported; the selected A02400 rat-brain IHC caption specifies 2.5 µg/ml but does not report a fixative (selected tissue-IHC caption). Antigen-retrieval dependence is unreported, so compare retrieved and unretrieved paraffin sections (standard IHC practice). The supplied evidence does not establish whether frozen sections or IF/ICC are easier; endogenous peroxidase in bone marrow can confound chromogenic staining (standard IHC practice).
HPA tissue IHC evidence for NUCB2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data. At least one protein variant secreted, tissue location of RNA and protein might differ and correlation is complex.). 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 |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Bronchus | Ciliated cells (cell body) | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Kidney | Cells in glomeruli | Not detected | Protein (IHC) | HPA → |
| Lung | Alveolar cells | Not detected | Protein (IHC) | HPA → |
| Ovary | Follicle cells | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Not detected | Protein (IHC) | HPA → |
| Urinary bladder | Urothelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NUCB2 IHC Tips
Troubleshoot NUCB2 chromogenic IHC in paraffin sections using compartment, cell type and antibody controls; use IF as a complementary localisation check.
How should I optimise retrieval when NUCB2 staining is weak in paraffin sections?
Start with Tris-EDTA at pH 9.0 for heat-induced epitope retrieval at 95–98 °C for 20 min (page retrieval setting). Run a known positive section, such as colon glandular cells, beside the test section to distinguish retrieval failure from low expression (HPA tissue IHC: High in colon glandular cells). If staining remains weak, compare a citrate buffer at pH 6.0 on adjacent sections while keeping antibody dilution and detection constant (standard IHC practice). Record both signal and tissue damage, because excessive heating can reduce interpretable morphology (standard IHC practice). Do not treat a changed staining pattern as evidence of a new NUCB2 compartment without antibody and tissue controls (UniProt P80303 localisation; standard IHC practice).
Could fixation explain variable NUCB2 staining between paraffin blocks?
NUCB2-specific sensitivity to fixation is unknown from the supplied evidence; the rat brain tissue-IHC caption for A02400 does not state a fixative (A02400 tissue-IHC caption). Compare adjacent sections from blocks with documented fixation histories using the same retrieval, antibody incubation and detection settings (standard IHC practice). Include a positive tissue control in each run, such as duodenal glandular cells, and inspect whether its cytoplasmic signal also varies (HPA tissue IHC: High in duodenal glandular cells; HPA tissue IHC: cytoplasmic expression in most tissues). If fixation history is missing, report it as unknown rather than assigning a NUCB2-specific cause to weak staining (standard IHC practice). Use preserved morphology and control staining together when deciding whether a block is suitable for scoring (standard IHC practice).
Where should NUCB2 staining appear, and when is nuclear staining suspicious?
Expect predominantly cytoplasmic staining across tissues, with a Golgi-associated pattern possible at higher resolution (HPA tissue IHC: cytoplasmic expression in most tissues; HPA subcellular: Golgi apparatus, enhanced). NUCB2 is also annotated in the endoplasmic reticulum, at the nuclear envelope and as secreted, so distinguish a perinuclear rim from staining throughout the nucleus (UniProt P80303 localisation). The protein has no transmembrane segment, making a sharp cell-surface outline alone insufficient evidence for specific NUCB2 detection (UniProt P80303 topology; standard IHC interpretation). Compare suspect staining with a positive control, a matched no-primary control and intact neighbouring cells (standard IHC practice). Score the compartment observed in the section rather than assigning an organelle from chromogenic resolution alone (standard IHC practice).
Can this antibody distinguish full-length NUCB2 from nesfatin-1 in tissue?
First map the catalog antibody’s stated immunogen or epitope against NUCB2 before interpreting chromogenic staining (standard IHC practice). NUCB2 has 2 listed isoforms and a processed nesfatin-1 chain spanning residues 25–106; the longer NUCB2 chain spans 25–420 (UniProt P80303 processing and isoforms). An epitope within residues 25–106 could detect material containing that shared sequence, whereas an epitope outside it would not directly identify isolated nesfatin-1 (UniProt P80303 processing; standard epitope logic). The supplied A02400 caption reports tissue staining but no epitope map or cleavage-specific validation (A02400 tissue-IHC caption). Report the result as NUCB2 immunoreactivity unless epitope mapping and suitable controls support a more specific molecular assignment (standard IHC practice).
How can IF help verify a puzzling NUCB2 chromogenic pattern?
Use IF as a complementary localisation check, pairing NUCB2 with a marker for the expected cell type, such as a glandular-cell marker when examining colon glands (HPA tissue IHC: High in colon glandular cells; standard IF practice). Choose spectrally separated fluorophores and place the weaker signal in a channel with low tissue autofluorescence, determined from an unstained section (standard IF practice). For an epitope expected in the cytoplasm or Golgi, optimise permeabilisation after fixation; an extracellular epitope requires access without assuming that intracellular permeabilisation is appropriate (HPA subcellular: Golgi apparatus; UniProt P80303 topology; standard IF practice). Acquire single-label and no-primary controls at identical exposure settings to check bleed-through and background (standard IF practice). Confirm that any fine Golgi-like IF pattern agrees with the broader cytoplasmic distribution assessed by IHC (HPA subcellular; HPA tissue IHC).
What should I check when NUCB2 IHC shows diffuse brown background?
Inspect a no-primary section first to separate detection-system background from antibody-dependent staining (standard IHC practice). For peroxidase-based chromogenic detection, verify the peroxidase block and wash steps, then compare shorter chromogen development and a more dilute primary antibody on adjacent sections (standard IHC practice). Check whether diffuse signal follows tissue folds, section edges or damaged regions instead of cell boundaries (standard IHC practice). NUCB2 is annotated as secreted as well as intracellular, so extracellular signal needs careful comparison with intact positive cells and controls before interpretation (UniProt P80303 localisation; standard IHC interpretation). Use the same counterstain and image settings across comparisons so a background change is visible (standard IHC practice).
How should I score NUCB2 staining across specimens with different cell compositions? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because NUCB2 tissue staining is broadly cytoplasmic and varies by cell type (HPA tissue IHC: cytoplasmic expression in most tissues; HPA tissue IHC: cell-specific levels). For intensity scoring, calculate an H-score as 1 × % weak + 2 × % moderate + 3 × % strong, using only the predefined cells (standard IHC practice). Alternatively, report % positive cells or positive-cell density per mm² within an annotated tissue region (standard IHC practice). Normalise counts to the number of eligible cells or measured viable tissue area, and exclude folds and necrosis consistently (standard IHC practice). Keep retrieval, detection, thresholds and imaging conditions constant across specimens, with a shared positive-control section in each run (standard IHC practice).
How can I distinguish true NUCB2 positivity from artefact in an unexpected cell population?
Check whether staining is mainly cytoplasmic and occurs in intact cells; a Golgi-associated pattern may support its localisation at finer resolution (HPA tissue IHC: cytoplasmic expression in most tissues; HPA subcellular: Golgi apparatus). Compare the cell population with reported tissue patterns: colon glandular cells show high staining, whereas glomerular cells are reported as not detected (HPA tissue IHC: colon glandular cells High; kidney glomerular cells Not detected). Treat staining confined to section edges, necrotic areas or the no-primary control as suspect, and check endogenous enzyme activity in the detection workflow (standard IHC practice). A diffuse nuclear-only result warrants review of antibody specificity and morphology, given the predominantly cytoplasmic tissue profile (HPA tissue IHC; standard IHC practice). Interpret a discordant result alongside positive and negative controls, recognising that secreted variants can complicate agreement between RNA and protein location (HPA tissue IHC reliability description).
Boster reagents
Best NUCB2 / Nucleobindin-2 IHC Antibodies
A02400 has IHC and IF images from rat brain tissue (catalog image captions); human, mouse and rat are listed as reactive species (catalog reactivity).
Real IHC data
Anti-Nucleobindin-2 NUCB2 Antibody
Cat # A02400
A02400 has an IHC image from rat brain tissue at 2.5 μg/mL (catalog IHC image caption). A02400 also has an IF image from rat brain tissue at 20 μg/mL (catalog IF image caption).
Which to pick: For tissue IHC, choose A02400: IHC-P is listed (catalog applications), its IHC image shows rat brain tissue (catalog IHC image caption), and the fixative is unreported (catalog IHC image caption). For IF/ICC, A02400 has rat brain IF data (catalog IF image caption); ICC validation is not reported (catalog applications). For work across species, A02400 lists human, mouse and rat reactivity (catalog reactivity), while its IHC and IF images document rat tissue only (catalog image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P80303 (NUCB2_HUMAN, Nucleobindin-2).
- Human Protein Atlas. NUCB2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. NUCB2 subcellular location (ICC-IF): Localized to the Golgi apparatus..
- Human Protein Atlas. NUCB2 antibody validation summary (3 antibodies).
- Prognostication of prostate cancer based on NUCB2 protein assessment: NUCB2 in prostate cancer. Journal of experimental & clinical cancer research : CR 2013 — PMC3852884.
- Nesfatin-1/Nucleobindin-2 Is a Potent Prognostic Marker and Enhances Cell Proliferation, Migration, and Invasion in Bladder Cancer. Disease markers 2018 — PMC6169241.
- High expression of NUCB2 promotes papillary thyroid cancer cells proliferation and invasion. OncoTargets and therapy 2019 — PMC6388962.
- High NUCB2 expression level represents an independent negative prognostic factor in Chinese cohorts of non-metastatic clear cell renal cell carcinoma patients. Oncotarget 2017 — PMC5471050.
- PubMed PMID:7811391 — UniProt-cited evidence.
- PubMed PMID:9639681 — UniProt-cited evidence.
- PubMed PMID:12087473 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NUCB2 (Nucleobindin-2). Expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A02400 at 1:100–1:500 (standard starting range) (the product's own IHC validation image is shown). Controls (HPA): positive Bone marrow (Hematopoietic cells); negative Kidney. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet A02400); 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-induced epitope retrieval in Tris-EDTA buffer, pH 9.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-NUCB2, 1:100–1:500 starting range (standard); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: A secreted variant can weaken RNA–protein concordance (HPA tissue IHC); Isoform / epitope: 2 isoforms; nesfatin-1 cleavage affects epitope choice (UniProt); Regulation: Expression regulation is not specified (UniProt). Evidence: UniProt P80303, HPA tissue IHC · Enhanced, HPA ICC-IF · Golgi apparatus., Datasheet A02400, PMC3852884, PMC6169241. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NUCB2/immunohistochemistry-ihc-guide.