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NOL3 / Nucleolar protein 3 · IHC design guide
Design Immunohistochemistry for NOL3
Plan chromogenic NOL3 IHC-P around the nuclear and cytoplasmic pattern reported in most tissues (HPA tissue IHC). Adrenal glandular cells are a high-staining reference and duodenal glandular cells an undetected comparator (HPA tissue IHC); the catalog antibody’s IHC starting concentration is 5 μg/mL (datasheet).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NOL3 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 | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Bone marrow+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining has medium consistency with RNA data (HPA tissue IHC) | |
| Regulation | Low tissue RNA specificity (HPA tissue RNA) | |
| Isoform / epitope | 3 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended NOL3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by a published ARC/NOL3 tissue IHC protocol (PMC3245742).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A03991); 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-NOL3, 5 μg/mL (datasheet A03991) |
| 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 | NOL3-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and nuclear expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA HIER at pH 9.0, 95–98 °C for 20 min (page antigen retrieval rule); the published protocol does not specify retrieval (PMC3245742).
Section 2
What Is the Expected NOL3 Staining Pattern?
For paraffin-section IHC, expect NOL3 staining in the cytoplasm and nucleus of many cell types, including strongly stained glandular and neuronal cells (HPA: tissue IHC). UniProt also places NOL3 in the nucleolus, mitochondrion and sarcoplasmic reticulum; it has no transmembrane segment (UniProt O60936). HPA rates tissue staining Enhanced, while noting medium agreement with RNA data and pending external verification (HPA: tissue IHC).
| Cytoplasmic and nuclear signal in adrenal, gallbladder, parathyroid or prostate glandular cells. | This fits the reported High staining in those specific cells (HPA: tissue IHC). Judge the signal against nearby tissue structure and controls; the reported pattern does not imply that every cell in the section must stain equally. |
| Predominantly crisp surface or extracellular staining, with little intracellular signal. | Question this as an artefact: UniProt lists intracellular locations and no transmembrane segment (UniProt O60936), while HPA describes cytoplasmic and nuclear tissue staining (HPA: tissue IHC). Recheck morphology and detection controls before assigning it to NOL3. |
| Strong signal mainly in cells reported as unstained, such as bone-marrow hematopoietic cells. | Treat the cell identity as a warning for cross-reactivity or endogenous detection activity, rather than calling the entire tissue negative (HPA: Not detected in bone-marrow hematopoietic cells; general IHC practice). Confirm the compartment and compare antibody-omission and detection controls. |
| Uniform brown haze across cells, stroma and empty spaces. | Diffuse signal that ignores cell boundaries is difficult to reconcile with HPA's cell-specific staining observations (HPA: tissue IHC). Assess background from blocking, detection reagents and chromogen development using appropriate controls (general IHC practice). |
| No signal in an intact section containing adrenal glandular cells. | A blank result conflicts with their reported High staining (HPA: adrenal glandular cells). First check that the intended cells and counterstain are visible; then review the IHC-P antibody and detection workflow (general IHC practice). |
💡Expected NOL3 appearanceA convincing positive shows intracellular cytoplasmic and nuclear staining, reaching High levels in the specified HPA-positive cells; dominant surface staining or uniform haze warrants a false-positive check (HPA: tissue IHC; UniProt O60936 topology; general IHC practice).
| Cell and compartment selection | HPA reports cytoplasmic and nuclear expression in most tissues, but assigns levels to named cell populations (HPA: tissue IHC). Score those cells separately; a negative stromal area does not negate a positive glandular population (general IHC practice). |
| Choice of positive and low-signal comparison | Adrenal glandular cells and cerebral-cortex neuronal cells are reported High; lung type I alveolar cells are Low (HPA: tissue IHC). Those observations support comparison, while the HPA reliability note limits certainty across specimens (HPA: Enhanced, pending external verification). |
| Antibody validation | CAB009208 has Enhanced IHC validation, meaning its pattern was reproduced by independent antibodies or orthogonal data (HPA: CAB009208 IHC Enhanced). The tissue profile still has medium staining-to-RNA consistency and awaits external verification (HPA: tissue IHC). |
| Protein distribution and topology | UniProt lists nucleolar, cytoplasmic, mitochondrial and sarcoplasmic-reticulum locations, and no transmembrane segment (UniProt O60936). These annotations make intracellular staining plausible; they do not establish that routine chromogenic IHC can resolve each organelle (general IHC practice). |
| Isoforms and processing | Three isoforms are listed, with a chain spanning residues 2–208 and no signal peptide or propeptide (UniProt O60936). Without a supplied epitope map, these facts cannot predict which isoforms CAB009208 detects or justify an extracellular-shedding interpretation. |
| IF/ICC Q: where is NOL3 seen? | A: HPA supports nucleoplasm and cytosol localization in ICC-IF and lists A-431, MCF-7 and U2OS images (HPA: subcellular; CAB009208 ICC Supported). Use the separate IF/ICC guide for that application; these images do not establish an IHC-P staining level. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive glandular cells are blank. | The sampled cells may differ from the named HPA-positive population, or the IHC detection run may have failed (HPA: adrenal glandular cells High; general IHC practice). | Confirm cell identity and section quality, then check the catalog antibody's IHC-P instructions and run controls (general IHC practice). No NOL3-specific fixation or retrieval sensitivity is established by the supplied sources. |
| Strong signal appears in a reported negative population. | Cross-reactivity or endogenous detection activity is possible; HPA reports Not detected for duodenal glandular cells, among other specific populations (HPA: tissue IHC; general IHC practice). | Verify the stained cells, review an antibody-omission control, and check the detection system's endogenous-activity block where applicable (general IHC practice). Do not treat a cell-level HPA call as a whole-tissue exclusion. |
| Only the tissue surface or extracellular material is brown. | That distribution conflicts with the reported cytoplasmic and nuclear profile and with the lack of a transmembrane segment (HPA: tissue IHC; UniProt O60936 topology). | Inspect section edges, morphology and detection controls for artefact; require an intracellular pattern in the expected cells before scoring NOL3 positive (general IHC practice; HPA: tissue IHC). |
| Nearly every structure has the same faint brown tint. | Diffuse background can obscure the cell-specific pattern described by HPA (HPA: tissue IHC; general IHC practice). | Compare the antibody-omission control, then review blocking, antibody concentration, washes and chromogen development using the IHC-P workflow (general IHC practice). Do not infer a NOL3-specific retrieval defect. |
| Nuclear signal is seen without obvious cytoplasmic signal, or the reverse. | HPA reports both compartments overall, while UniProt lists several intracellular locations (HPA: tissue IHC; UniProt O60936). Neither source requires identical compartment intensity in every cell. | Score nuclear and cytoplasmic signal separately in identified cells, compare a known HPA-positive population, and avoid calling one compartment alone definitive (general IHC practice). |
| A muscle-rich specimen is weaker than expected. | UniProt reports high expression in heart and skeletal muscle, whereas the supplied HPA IHC table marks smooth-muscle cells Not detected (UniProt O60936; HPA: smooth muscle). These are different muscle populations and evidence types. | Identify the muscle population before interpreting the result; use the HPA cell-specific IHC observations for slide comparison and report any discrepancy without assigning a fixation cause. |
Experiment evidence
IHC & IF images showing NOL3 staining
A03991 · IHCImmunohistochemistry of ARC in human heart tissue with ARC antibody at 5 μg/mL.
A03991 · IHCImmunohistochemistry of ARC in human skeletal muscle with ARC antibody at 5 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000140939-NOL3/tissue/cerebral+cortex#imid_2856516
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000140939-NOL3/tissue/colon#imid_2856458
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000140939-NOL3/tissue/liver#imid_2856443
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000140939-NOL3/tissue/kidney#imid_2856460
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB009208; green = NOL3, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB009208; green = NOL3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NOL3 IHC & IF
🧪Run adrenal gland first: glandular cells should stain (HPA: High in adrenal gland glandular cells). Use bone marrow hematopoietic cells as the negative tissue (HPA: Not detected); on the adrenal slide, use any nonglandular cells without specific staining as internal background comparators, without assuming those cells are NOL3-negative.
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Bone marrow (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show NOL3 in A-431, MCF-7, U2OS, with annotated localisation: Nucleoplasm (supported), Cytosol (supported) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, a nonimmune immunoglobulin control matched to the primary antibody’s host species and monoclonal or polyclonal format, and NOL3 knockout material or peptide competition if the immunizing peptide is available (standard IHC practice). On adrenal sections, check endogenous peroxidase background; check endogenous biotin if using avidin–biotin detection, or autofluorescence if using fluorescence detection (standard IHC/IF practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the A03991 heart-tissue IHC caption does not state a fixative (selected-SKU caption). NOL3 retrieval dependency is unreported; compare retrieved and unretrieved paraffin sections (standard IHC practice). The supplied evidence does not establish whether frozen sections or IF are easier; adrenal background should be checked against the controls above, while ICC-IF images support nucleoplasm and cytosol localization in A-431, MCF-7 and U2OS (HPA: subcellular).
HPA tissue IHC evidence for NOL3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data. Pending external verification.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Parathyroid gland | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Ovary | Ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Small intestine | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NOL3 IHC Tips
Troubleshoot NOL3 staining in paraffin sections by checking retrieval, compartment-specific signal, controls, and scoring before interpreting chromogenic IHC results.
What retrieval should I start with for weak NOL3 staining in paraffin sections?
Use Tris-EDTA pH 9.0 HIER at 95–98 °C for 20 min for paraffin-section NOL3 IHC (page retrieval rule: nuclear antigen). Let sections cool in retrieval buffer, then compare the weak section with a concurrently processed positive control to separate retrieval failure from detection failure (standard IHC practice). If staining remains weak, trial citrate pH 6.0 on matched sections as a fallback and compare signal with tissue preservation before changing another variable (standard IHC practice). Score nuclear and cytoplasmic signal separately during that comparison, because both compartments are plausible for NOL3 (HPA subcellular: nucleoplasm and cytosol supported; HPA tissue IHC: cytoplasmic and nuclear expression).
Could fixation explain inconsistent NOL3 staining across paraffin blocks?
NOL3-specific fixation sensitivity is unknown because no target-specific fixation evidence is supplied for this guide (supplied evidence: no target-specific fixation data). Record each block’s fixative and fixation interval before comparing staining, since fixation differences can change epitope accessibility in routine IHC (standard IHC practice). For a prospective comparison, process matched samples in 10% neutral buffered formalin near pH 7.0 and hold retrieval and detection conditions constant (standard IHC practice). Assess morphology alongside signal, and repeat a discordant block with the page’s Tris-EDTA pH 9.0, 20 min retrieval before assigning a biological difference (page retrieval rule; standard IHC practice).
Should NOL3 stain the nucleus, cytoplasm, or both?
Expect to evaluate both nuclear and cytoplasmic staining: tissue IHC reports both, while cell imaging supports nucleoplasm and cytosol (HPA tissue IHC: cytoplasmic and nuclear expression; HPA subcellular: supported locations). Nucleolar, mitochondrial, and sarcoplasmic-reticulum associations are also listed, but a chromogenic section cannot establish those fine compartments from color alone (UniProt O60936 subcellular; standard IHC interpretation). Record nuclear and cytoplasmic scores separately rather than treating one compartment as nonspecific by default (HPA tissue IHC; standard IHC practice). If a shift appears after retrieval changes, compare serial sections processed for 20 min under the page conditions before interpreting redistribution (page retrieval rule; standard IHC practice).
How could isoforms or epitope location alter my NOL3 IHC pattern?
NOL3 has 3 listed isoforms, but the supplied tissue-IHC caption does not identify the antibody’s epitope or establish isoform coverage (UniProt O60936 isoforms; selected A03991 caption). Its CARD spans residues 4–95, phosphorylation is reported at Thr149, and the serine-rich C-terminus mediates nuclear localization (UniProt O60936 domains, modified residues, subcellular note). Those features make epitope placement worth checking in antibody documentation; they do not show that this antibody distinguishes an isoform or phosphorylation state (UniProt O60936; selected A03991 caption). When patterns disagree, compare compartment scores and a matched positive control under the same retrieval and detection conditions before attributing the difference to an isoform (standard IHC practice).
How can parallel IF help assess an uncertain NOL3 IHC pattern?
Use parallel IF/ICC to inspect nucleoplasm and cytosol more clearly than a chromogenic section permits (HPA subcellular: both locations supported; standard microscopy practice). Multiplex NOL3 with a validated marker for the cell type being assessed, such as the relevant glandular or neuronal population, and include single-stain controls to test channel bleed-through (HPA tissue IHC: glandular and neuronal staining; standard IF practice). Choose a longer-wavelength fluorophore when tissue autofluorescence obscures shorter-wavelength signal, checking an unstained sample first (standard IF practice). For intracellular nuclear and cytosolic epitopes, assess permeabilisation on matched samples; NOL3 has no transmembrane segment, but this antibody’s epitope is unspecified (UniProt O60936 topology; selected A03991 caption; standard IF practice).
What should I check when NOL3 DAB staining looks diffuse?
Run a no-primary control through the same detection steps to identify signal from the detection system or tissue rather than antibody binding (standard chromogenic IHC practice). Apply a peroxidase block before DAB detection, and inspect the control for residual endogenous enzyme activity (standard chromogenic IHC practice). Check whether the catalog antibody concentration matches the documented heart-tissue image, 5 µg/mL, while recognising that the caption does not establish an optimum for every section (selected A03991 caption). Compare stained and control sections at equal development times, then adjust blocking or antibody concentration one variable at a time while preserving tissue morphology (standard IHC practice).
How should I score NOL3 across mixed cell populations? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and viable area before scoring, and keep nuclear and cytoplasmic staining as separate results (HPA tissue IHC: both compartments; standard IHC scoring practice). For each compartment, record the percentage of positive target cells and intensity on a 0–3 scale; an H-score is the sum of intensity-weighted percentages, ranging from 0–300 (standard IHC scoring practice). In mixed tissue, report positive-cell density per mm² of viable target area when cell counting is unreliable (standard image-analysis practice). Normalise comparisons to the same cell type and sampled area, with identical thresholds and detection settings across slides (standard IHC quantification practice).
How do I distinguish credible NOL3 signal from an IHC artefact?
Look for reproducible nuclear or cytoplasmic staining in intact cells, consistent with the reported tissue pattern, rather than relying on a single dark region (HPA tissue IHC: cytoplasmic and nuclear expression; standard IHC interpretation). Human heart has a documented antibody-stained image at 5 µg/mL, while glandular cells in adrenal gland and neuronal cells in cerebral cortex are reported high by tissue IHC (selected A03991 caption; HPA tissue IHC). Treat staining confined to section edges, necrotic regions, or the no-primary control as suspect, and check peroxidase blocking for DAB signal (standard chromogenic IHC practice). Compare the same cell type across sections before calling an unexpected compartment or positive population biological; the HPA tissue profile remains pending external verification (HPA tissue IHC reliability; standard IHC interpretation).
Boster reagents
Best NOL3 / Nucleolar protein 3 IHC Antibodies
The IHC-validated NOL3 antibody has human heart and skeletal muscle IHC images (A03991 image captions). No IF image is provided (catalog images).
Real IHC data
Anti-ARC NOL3 Antibody
Cat # A03991
A03991 is listed for human IHC-P (catalog applications and reactivity). Its IHC images show human heart and skeletal muscle at 5 μg/mL (A03991 image captions).
Which to pick: Choose A03991 for human paraffin-section IHC (catalog applications: IHC-P; A03991 image captions: human heart and skeletal muscle); its image captions do not report the fixative. A03991 is rabbit-hosted, but its clonality is unreported (catalog host and clone fields). No listed SKU is validated for IF/ICC or another species (catalog applications, reactivity and IF images).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O60936 (NOL3_HUMAN, Nucleolar protein 3).
- Human Protein Atlas. NOL3 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. NOL3 subcellular location (ICC-IF): Localized to the nucleoplasm and cytosol..
- Human Protein Atlas. NOL3 antibody validation summary (1 antibodies).
- Molecular Characterization and Clinical Relevance of RNA Binding Proteins in Colorectal Cancer. Frontiers in genetics 2020 — PMC7597397.
- Apoptosis inhibitor ARC promotes breast tumorigenesis, metastasis, and chemoresistance. Cancer research 2011 — PMC3245742.
- The Cell Death Inhibitor ARC Is Induced in a Tissue-Specific Manner by Deletion of the Tumor Suppressor Gene Men1, but Not Required for Tumor Development and Growth. PloS one 2015 — PMC4692498.
- PubMed PMID:9560245 — UniProt-cited evidence.
- PubMed PMID:10196175 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NOL3 (Nucleolar protein 3). Expected localisation Nuclear and cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A03991 at 5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Bone marrow. 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 A03991); 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-NOL3, 5 μg/mL (datasheet A03991); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining has medium consistency with RNA data (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope coverage is unspecified (UniProt); Regulation: Low tissue RNA specificity (HPA tissue RNA). Evidence: UniProt O60936, HPA tissue IHC · Enhanced, HPA ICC-IF · nucleoplasm and cytosol., Datasheet A03991, PMC3245742. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NOL3/immunohistochemistry-ihc-guide.