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NPAS3 / Neuronal PAS domain-containing protein 3 · IHC design guide
Design Immunohistochemistry for NPAS3
Plan NPAS3 IHC-P around nuclear staining in caudate neurons and cerebral cortex glia (HPA tissue IHC). This guide covers fixation, retrieval, controls and scoring; start catalog antibody A10668 at 5 μg/mL (datasheet).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NPAS3 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 in tissue sections (HPA tissue IHC) | |
| Staining pattern | Nuclear staining in most tissues, strongest in CNS (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Caudate+4 more · see all | |
| Negative control | Adipose tissue+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 | Staining may disagree with RNA levels (HPA tissue IHC) | |
| Regulation | Widespread expression in adult brain (UniProt) | |
| Isoform / epitope | 6 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended NPAS3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by one published NPAS3 staining method for human TNBC tissue (PMC11379032).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A10668); 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-NPAS3, 5 μg/mL (datasheet A10668) |
| 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 | NPAS3-positive staining in neuronal cells of caudate (HPA tissue IHC: High). HPA tissue profile: Nuclear expression in a majority of tissues, most abundant in CNS. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 retrieval at 95–98 °C for 20 min (page retrieval rule; UniProt Q8IXF0: nuclear localisation).
Section 2
What Is the Expected NPAS3 Staining Pattern?
NPAS3 is a nuclear protein with no transmembrane segment (UniProt Q8IXF0: subcellular location and topology). In paraffin sections, expect nuclear staining across many tissues, strongest in CNS examples: caudate neuronal cells and cerebral cortex glial cells (HPA tissue IHC: High). HPA rates the tissue profile Enhanced but reports low consistency between antibody staining and RNA expression; interpret unexpected staining with that limitation in mind (HPA tissue IHC).
| Distinct nuclear staining in caudate neuronal cells or cerebral cortex glial cells (HPA tissue IHC: High). | This matches the strongest listed cellular patterns and the expected nuclear compartment (HPA tissue IHC; UniProt Q8IXF0: Nucleus). Score the named cell population, its nuclear intensity and the proportion stained; do not treat every cell in the section as equivalent (general IHC practice). |
| Predominantly cytoplasmic or membranous color, with little nuclear signal (UniProt Q8IXF0: Nucleus; HPA tissue IHC: nuclear profile). | That distribution conflicts with the reported location and should be treated as suspect (UniProt Q8IXF0; HPA tissue IHC). Review morphology and the detection controls before assigning NPAS3 positivity; chromogen precipitate or nonspecific staining can obscure the compartment (general IHC practice). |
| Strong staining in adipocytes or bone marrow hematopoietic cells (HPA tissue IHC: Not detected). | These cell populations are useful low-signal comparators, although an HPA nondetection is not proof of biological absence (HPA tissue IHC). If they stain while the expected nuclei do not, investigate cross-reactivity, endogenous detection activity and cell identification (general IHC practice). |
| Broad haze, edge staining or color over several compartments, including areas outside identifiable cells (general IHC practice). | Diffuse background prevents a reliable nuclear score (general IHC practice). Compare a reagent-control section and inspect washing, blocking, antibody concentration and chromogen development before changing the biological interpretation (general IHC practice). |
| No nuclear signal in caudate neuronal cells or cerebral cortex glial cells (HPA tissue IHC: High). | First consider a failed run or an unsuitable section, while allowing for sample variation (general IHC practice). Confirm that nuclei and tissue morphology are preserved, then check retrieval, antibody dilution and detection with an independent positive control (general IHC practice). |
💡Expected NPAS3 appearanceCall a result positive when chromogen is concentrated in nuclei of identifiable caudate neuronal cells or cerebral cortex glial cells at the reported High level (HPA tissue IHC); predominantly cytoplasmic color or strong staining of HPA-listed nondetected cell populations is a warning to investigate, not an NPAS3 call (UniProt Q8IXF0: Nucleus; HPA tissue IHC).
| Cell population and tissue context (HPA tissue IHC). | Caudate neuronal cells and cerebral cortex glial cells are listed High; several glandular or epithelial populations are Medium, while cerebellar molecular-layer cells are Low (HPA tissue IHC). Choose comparators by the named cell population rather than by an entire organ's label (general IHC practice). |
| Tissue-profile reliability (HPA tissue IHC: Enhanced). | HPA also reports low consistency between antibody staining and RNA expression (HPA tissue IHC). Treat the observed IHC pattern as a guide, and corroborate a surprising result with controls and cell-level morphology (general IHC practice). |
| Antibody validation scope (HPA antibodies: HPA075337). | The listed rabbit polyclonal antibody is Approved for IHC and Supported for ICC (HPA antibodies: HPA075337). Those labels do not supply a section-specific dilution or guarantee that an unexpected compartment represents NPAS3 (HPA antibodies; general IHC practice). |
| Isoforms and epitope coverage (UniProt Q8IXF0: 6 isoforms). | Six isoforms are listed, but the supplied record does not map the IHC antibody's epitope to them (UniProt Q8IXF0; HPA antibodies). Do not infer which isoforms contribute to staining or explain a negative section by isoform loss without separate evidence. |
| Protein location and processing (UniProt Q8IXF0). | NPAS3 is nuclear, has no transmembrane segment and has no listed signal peptide or propeptide (UniProt Q8IXF0). These annotations support nuclear scoring; they do not establish an NPAS3-specific retrieval condition or fixation sensitivity. |
| IF/ICC cross-check (HPA subcellular; HPA antibodies: HPA075337). | Q: What should an IF/ICC image show? A: Nucleoplasmic signal is the supported HPA location, and the listed antibody has Supported ICC status (HPA subcellular; HPA antibodies). Use that as a localization check; assess IF/ICC methods in its separate guide. |
| Situation | Likely cause | Next action |
|---|---|---|
| CNS positive control has no nuclear staining (HPA tissue IHC: caudate or cerebral cortex High). | Section quality, retrieval or detection may have failed; a blank slide alone cannot identify which step failed (general IHC practice). | Inspect tissue morphology and counterstain, then verify retrieval, antibody dilution and detection with a working positive control (general IHC practice). Do not label the test specimen negative until the run performs. |
| Signal appears mainly in cytoplasm or on cell borders (UniProt Q8IXF0: Nucleus). | The compartment conflicts with UniProt's nuclear annotation and HPA's nuclear tissue profile (UniProt Q8IXF0; HPA tissue IHC). Background or misread cell borders are possible (general IHC practice). | Recheck nuclear boundaries on the counterstained section and compare reagent controls (general IHC practice). Score only convincing cellular nuclear signal, and flag persistent nonnuclear staining for validation. |
| Adipocytes or marrow hematopoietic cells stain strongly (HPA tissue IHC: Not detected). | The result differs from the listed HPA cell-level pattern; cross-reactivity or endogenous detection activity is possible, not established (HPA tissue IHC; general IHC practice). | Identify the stained cells morphologically and use appropriate reagent and endogenous-activity controls (general IHC practice). Compare with a known-positive nuclear pattern before interpreting the unusual signal. |
| Most of the section has diffuse chromogen that hides nuclei (general IHC practice). | Excess background can arise from antibody concentration, incomplete washing, blocking or detection development (general IHC practice). The appearance alone cannot assign one cause. | Review the reagent-control section; optimize concentration, washing, blocking or development one variable at a time (general IHC practice). Re-score only when nuclei can be distinguished from background. |
| A glandular region is negative despite tissue-level RNA interest (HPA tissue IHC: cervix glandular cells Not detected; RNA tissue enhanced). | RNA enrichment does not establish detectable protein staining in that cell population; the supplied HPA observations differ (HPA tissue IHC). | Report the cell-level IHC observation separately from the RNA annotation (HPA tissue IHC). Confirm run performance in a listed High CNS population before interpreting a negative result (general IHC practice). |
| An IF/ICC result is used to judge a paraffin IHC section (HPA subcellular; HPA antibodies). | HPA supports nucleoplasmic ICC localization, while the listed antibody's IHC and ICC validation labels describe separate applications (HPA subcellular; HPA antibodies: HPA075337). | Use the IF/ICC image to check the expected compartment, then judge the paraffin section against its own cell-level IHC pattern and controls (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing NPAS3 staining
A10668 · IHCImmunohistochemistry of NPAS3 in human brain tissue with NPAS3 antibody at 5 μg/mL.
A10668 · IFImmunofluorescence of NPAS3 in human brain tissue with NPAS3 antibody at 20 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000151322-NPAS3/tissue/cerebral+cortex#imid_20844215
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000151322-NPAS3/tissue/colon#imid_20844125
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000151322-NPAS3/tissue/liver#imid_20844142
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000151322-NPAS3/tissue/kidney#imid_20844127
HPA · ICC-IF AF22Human Protein Atlas immunofluorescence, AF22, antibody HPA075337; green = NPAS3, blue = nucleus, red = microtubules.
HPA · ICC-IF AF22Human Protein Atlas immunofluorescence, AF22, antibody HPA075337; green = NPAS3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NPAS3 IHC & IF
🧪Run caudate first and expect staining in neuronal cells (HPA: High in caudate neuronal cells). Use adipose tissue as the negative tissue (HPA: Not detected in adipocytes); on the caudate slide, neighboring non-neuronal cells should show only background signal while neuronal nuclei are evaluated (UniProt Q8IXF0: nucleus).
Positive control tissue: Caudate (Neuronal cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show NPAS3 in AF22, PC-3, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, an isotype control matched to the primary antibody’s host species and clonality, and NPAS3-knockout tissue if available. For chromogenic caudate IHC, block endogenous peroxidase and check for brain pigment that could be mistaken for signal (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and retrieval dependency are unreported in the supplied evidence; the selected A10668 brain IHC caption reports 5 μg/mL but does not state a fixative (selected A10668 caption). Optimize antigen retrieval on paraffin sections, then assess nuclear staining (UniProt Q8IXF0: nucleus). The supplied evidence does not establish that frozen sections or IF are easier; brain autofluorescence can complicate IF assessment (standard IF practice).
HPA tissue IHC evidence for NPAS3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Low consistency between antibody staining and RNA expression data.). 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 |
|---|---|---|---|---|
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Glial cells | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NPAS3 IHC Tips
Troubleshoot NPAS3 staining in paraffin sections by checking nuclear localisation, tissue controls, and processing conditions before comparing signal intensity.
What should I change if NPAS3 nuclear staining is weak after retrieval?
Start with Tris-EDTA at pH 9.0 for HIER at 95–98 °C for 20 min (page retrieval specification). Compare a matched positive control on the same run, because NPAS3 is reported in nuclei and is abundant in CNS tissue (UniProt: nucleus; HPA: nuclear expression, most abundant in CNS). If signal remains weak, test a shorter or longer heating interval on adjacent sections while holding antibody concentration and detection constant (standard IHC practice). Record tissue integrity and nuclear background alongside signal; excessive retrieval can compromise morphology and make a faint nuclear result difficult to interpret (standard IHC practice).
Could fixation explain weak NPAS3 staining in my paraffin sections?
NPAS3-specific sensitivity to fixation is unknown from the supplied evidence; the selected tissue-IHC caption does not state a fixative (A10668 caption: fixative not stated). Compare sections with documented fixation histories while keeping retrieval at Tris-EDTA pH 9.0, 95–98 °C for 20 min and using one detection run (page retrieval specification; standard IHC practice). Include a known positive tissue control and assess nuclear morphology before interpreting a weak result as biological absence (HPA: high in caudate neuronal cells; standard IHC practice). Do not infer a fixation response from NPAS3’s lack of a transmembrane segment or annotated glycosylation sites (UniProt: topology and glycosylation).
How should I assess cytoplasmic staining when NPAS3 is expected in nuclei?
Prioritise discrete nuclear staining: NPAS3 is annotated in the nucleus, and its supported subcellular location is the nucleoplasm (UniProt: nucleus; HPA: nucleoplasm supported). Check whether the signal follows intact nuclei across the section, using the counterstain to identify boundaries and exclude folds or damaged areas (standard IHC practice). If cytoplasmic colour dominates, compare an antibody omission control and a matched positive control before assigning it to NPAS3 (standard IHC practice; HPA: high in caudate neuronal cells). Score nuclear and cytoplasmic compartments separately, because a combined intensity score would obscure the localisation discrepancy (UniProt: nucleus; standard IHC practice).
Can an epitope difference explain discordant NPAS3 staining?
NPAS3 has 6 listed isoforms, so an antibody’s epitope matters when comparing staining across samples or assays (UniProt: isoforms 1–6; standard IHC practice). Check the catalog antibody’s documented immunogen or epitope against the relevant isoforms before interpreting a negative nucleus as absence of all NPAS3 forms (UniProt: isoforms 1–6; standard IHC practice). Its annotated bHLH region spans residues 51–104, with PAS regions at 147–217 and 319–389; these coordinates do not establish where this antibody binds (UniProt: domains). If the epitope is undisclosed, report that limitation and compare an independently validated antibody with a known epitope on adjacent sections (standard IHC practice).
How can I check an NPAS3 IHC result with multiplex immunofluorescence?
Use a separately validated IF/ICC workflow and pair NPAS3 with a marker for the expected neuronal or glial population in the tissue being examined (HPA: high in caudate neuronal cells and cerebral cortex glial cells; standard IF practice). Choose spectrally separated fluorophores and inspect unstained tissue for autofluorescence before assigning a weak nuclear signal, especially in brain sections (standard IF practice). NPAS3 has no transmembrane segment and is annotated as nuclear, so use permeabilisation appropriate for access to an intracellular nuclear epitope (UniProt: topology and subcellular location; standard IF practice). Confirm colocalisation within nuclear boundaries using a nuclear counterstain and single-colour controls (HPA: nucleoplasm supported; standard IF practice).
What controls distinguish NPAS3 staining from chromogenic background?
Run an antibody omission control to identify signal from the detection system, and compare it with an expected positive tissue section processed in the same batch (standard IHC practice; HPA: high in caudate neuronal cells). For peroxidase detection, check that the peroxidase block is effective and that DAB development is stopped consistently across slides (standard chromogenic IHC practice). Examine diffuse colour, section edges, folds, and damaged regions separately from intact nuclei before adjusting antibody concentration (standard IHC practice; HPA: nucleoplasm supported). If background persists, compare blocking and primary antibody titration on adjacent sections; the selected image caption reports 5 µg/mL, without establishing an optimum for every specimen (A10668 caption; standard IHC practice).
How should I quantify NPAS3 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and anatomical region before scoring, because HPA reports high NPAS3 staining in caudate neuronal cells and cerebral cortex glial cells (HPA: tissue IHC profile; standard IHC practice). Report the percentage of positive nuclei and a nuclear H-score using intensity categories 0–3, or report positive nuclei per mm² when cell density differs (standard IHC quantification practice). Normalise each result to counted nuclei or measured tissue area, and apply the same threshold, counterstain review, and imaging settings across samples (standard IHC practice). Exclude folds and damaged tissue from the analysed area and report these exclusions with the scoring method (standard IHC practice).
When is an apparent NPAS3-positive cell convincing rather than artefactual?
A convincing result is nuclear signal within intact cells in an appropriate tissue context; NPAS3 is nuclear, and HPA reports prominent CNS staining (UniProt: nucleus; HPA: nuclear expression, most abundant in CNS). Check whether the stained cells match the population being scored, such as caudate neurons or cerebral cortex glia, before calling a tissue positive (HPA: high in those cell populations). Discount isolated edge staining, necrotic areas, and diffuse cytoplasmic colour, and review the antibody omission control for endogenous enzyme or detection background (standard IHC practice; HPA: nucleoplasm supported). Interpret negative samples cautiously because HPA reports low consistency between antibody staining and RNA expression data (HPA: reliability description).
Boster reagents
Best NPAS3 / Neuronal PAS domain-containing protein 3 IHC Antibodies
A10668 is an IHC-P and IF anti-NPAS3 antibody (catalog: applications) with human brain tissue images for both methods (catalog: image captions); Human, Mouse and Rat are listed as reactive (catalog: reactivity).
Real IHC data
Anti-NPAS3 Antibody
Cat # A10668
A10668 will render with an IHC image of human brain tissue at 5 μg/mL (catalog: IHC image caption). Its IF image shows human brain tissue at 20 μg/mL (catalog: IF image caption); the catalog lists Human, Mouse and Rat reactivity (catalog: reactivity).
Which to pick: Choose A10668 for paraffin-section IHC (catalog: IHC-P application), using 5 μg/mL as the starting concentration shown in its human brain image (catalog: IHC image caption); the fixative is unreported (catalog: IHC image caption). Choose A10668 for tissue IF at the pictured 20 μg/mL (catalog: IF image caption); ICC validation is not documented (catalog: applications and image captions). For cross-species work, A10668 lists Human, Mouse and Rat reactivity (catalog: reactivity), while its supplied IHC and IF images show human tissue only (catalog: image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8IXF0 (NPAS3_HUMAN, Neuronal PAS domain-containing protein 3).
- Human Protein Atlas. NPAS3 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. NPAS3 subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. NPAS3 antibody validation summary (1 antibodies).
- A Prognostic Risk Signature of Two Autophagy-Related Genes for Predicting Triple-Negative Breast Cancer Outcomes. Breast cancer (Dove Medical Press) 2024 — PMC11379032.
- PubMed PMID:12746393 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NPAS3 (Neuronal PAS domain-containing protein 3). Expected localisation Nuclear in tissue sections (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A10668 at 5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Caudate (Neuronal cells); negative Adipose tissue. 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 A10668); 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-NPAS3, 5 μg/mL (datasheet A10668); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining may disagree with RNA levels (HPA tissue IHC); Isoform / epitope: 6 isoforms; epitope coverage is unspecified (UniProt); Regulation: Widespread expression in adult brain (UniProt). Evidence: UniProt Q8IXF0, HPA tissue IHC · Enhanced, HPA ICC-IF · nucleoplasm., Datasheet A10668, PMC11379032. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NPAS3/immunohistochemistry-ihc-guide.