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NOS1AP / Carboxyl-terminal PDZ ligand of neuronal nitric oxide synthase protein · IHC design guide
Design Immunohistochemistry for NOS1AP
Plan chromogenic NOS1AP IHC in paraffin sections using the catalog antibody’s 2 μg/ml example (datasheet A03060-3). Compare neuronal cytoplasmic staining with the HPA tissue pattern and assess suspected off-target signal before scoring (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NOS1AP 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 | Neuronal cytoplasm observed (HPA tissue IHC); filopodia and podosomes annotated (UniProt) | |
| Staining pattern | Cytoplasmic staining in neuronal cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A03060-3) | |
| 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 | Presumed off-target binding may confound scoring (HPA tissue IHC) | |
| Regulation | Expression varies by tissue (HPA tissue IHC) | |
| Isoform / epitope | 3 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended NOS1AP IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with the published breast cancer IHC protocol (PMC3419983).
| Sample | Paraffin-embedded mouse brain tissue; fixative not specified (datasheet A03060-3) |
| Fixation | Image fixative and duration unreported (datasheet A03060-3); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A03060-3); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A03060-3) |
| Primary antibody | Rabbit anti-NOS1AP, 2-5 μg/ml (datasheet A03060-3) |
| Primary incubation | Overnight at 4 °C (datasheet A03060-3) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A03060-3) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | NOS1AP-positive staining in neuronal cells of caudate (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in neuronal cells. No signal in the no-primary control. |
💡Decision noteTry heat-mediated EDTA retrieval at pH 8.0 first; both the catalog datasheet and published protocol specify it (datasheet A03060-3; PMC3419983).
Section 2
What Is the Expected NOS1AP Staining Pattern?
In paraffin-section IHC, expect NOS1AP mainly in neuronal cytoplasm, with Medium staining reported in caudate, cerebral cortex, and hippocampus (HPA tissue IHC: Approved). UniProt also places NOS1AP in filopodia and podosomes and reports no transmembrane segment (UniProt O75052). Interpret this pattern cautiously: HPA reports low agreement between antibody staining and RNA expression and disregarded presumed off-target staining (HPA tissue IHC: reliability description).
| Neuronal cell bodies show cytoplasmic chromogen in caudate, cortex, or hippocampus. | This fits HPA's observed Medium neuronal staining and cytoplasmic profile (HPA tissue IHC). Judge the labeled cells and compartment together; intensity alone cannot establish specificity, given HPA's reported RNA–staining inconsistency and presumed off-target binding (HPA tissue IHC: reliability description). |
| Brain sections show only nuclear staining, or a sharp outline confined to the plasma membrane. | That differs from HPA's neuronal cytoplasmic IHC profile (HPA tissue IHC). A membrane-only interpretation is also unsupported by the absence of a transmembrane segment (UniProt O75052). Nuclear staining needs an assay-specific check: nucleoplasm is an approved ICC-IF location, so it is not automatically an artefact in IF (HPA subcellular). |
| Strong staining appears across cells or tissues listed as undetected. | Check whether the staining is confined to the cell type HPA actually scored: adipocytes in adipose tissue and glandular cells in adrenal gland were Not detected (HPA tissue IHC). Widespread signal in those scored cells raises concern for nonspecific binding or endogenous chromogen activity (general IHC practice); the HPA reliability note strengthens the need for controls. |
| Chromogen forms a diffuse haze over tissue, including areas without identifiable labeled cells. | An indistinct field cannot support the expected neuronal cytoplasmic call (HPA tissue IHC: profile). Consider excess primary or detection reagent, inadequate washing, or endogenous enzyme activity, and assess a matched no-primary section (general chromogenic IHC practice). Do not score haze as NOS1AP-positive cells. |
| No neuronal signal is visible in a caudate, cortex, or hippocampus section. | Those tissues contain HPA-scored Medium neuronal staining (HPA tissue IHC), so first check that recognizable neuronal cells and an interpretable counterstain are present. A failed run, unsuitable retrieval conditions, or weak detection can also erase an expected pattern (general IHC practice); one negative section does not establish biological absence. |
💡Expected NOS1AP appearanceCall a positive IHC result when identifiable neuronal cells show mainly cytoplasmic, approximately Medium chromogen in HPA-positive brain tissue; diffuse haze or widespread staining in HPA-undetected cell types warrants a specificity check (HPA tissue IHC; general chromogenic IHC practice).
| Reference tissue and scored cell type | Caudate, cortex, and hippocampus provide observed Medium neuronal examples; HPA also reports Medium staining in fallopian-tube ciliated cell bodies and testis Leydig cells (HPA tissue IHC). Compare like cell types rather than treating every cell in a positive tissue as positive. |
| Location depends on assay | HPA describes neuronal cytoplasm by tissue IHC, but approves nucleoplasm and vesicles by ICC-IF (HPA tissue IHC; HPA subcellular). UniProt adds filopodia and podosomes (UniProt O75052). Keep those observations tied to their assay and context when judging an apparent compartment mismatch. |
| Antibody evidence | Three listed rabbit antibodies have IHC status Approved; only HPA030066 also has ICC status Approved (HPA antibodies). HPA still reports low staining–RNA consistency and presumed off-target binding for the tissue profile (HPA tissue IHC). An Approved status does not replace slide controls. |
| Isoforms and epitope coverage | UniProt lists 3 NOS1AP isoforms and a PID domain at residues 26–196 (UniProt O75052). The supplied record does not map an antibody epitope or show which isoforms it detects, so isoform-specific staining predictions would be unsupported. |
| Processing and topology | The annotated chain spans residues 1–506, with no signal peptide, propeptide, or transmembrane segment (UniProt O75052). These annotations support caution about a membrane-only or shed-protein explanation; they do not establish an antigen-retrieval condition or fixation sensitivity. |
| Situation | Likely cause | Next action |
|---|---|---|
| Brain reference tissue has little or no neuronal staining. | The run may have inadequate detection, or the chosen retrieval conditions may perform poorly (general IHC practice); HPA reports Medium neuronal staining in caudate, cortex, and hippocampus (HPA tissue IHC). | Confirm the section contains identifiable neurons, check the run's positive control and reagent steps, then optimize retrieval and antibody concentration within the laboratory's IHC workflow (general IHC practice). Reassess the same cell type rather than increasing exposure until background appears. |
| The chromogen is predominantly nuclear in neuronal cells. | A nuclear-only tissue IHC pattern conflicts with HPA's neuronal cytoplasmic profile (HPA tissue IHC), although nucleoplasm is an approved ICC-IF location (HPA subcellular). The two assays should not be assigned one universal compartment rule. | Check the counterstain and focus plane, compare a matched no-primary control, and repeat with a validated IHC antibody if the pattern persists (general IHC practice; HPA antibodies: IHC Approved). Report the compartment and assay explicitly. |
| Adipose adipocytes or adrenal glandular cells appear broadly positive. | HPA scores those particular cells Not detected and notes presumed off-target binding in its tissue assessment (HPA tissue IHC). Endogenous detection activity can also produce chromogen without specific primary binding (general chromogenic IHC practice). | Run no-primary and detection-only controls, block the endogenous activity relevant to the chosen chromogen system, and compare staining with the scored cell types on HPA-positive sections (general IHC practice; HPA tissue IHC). |
| Signal is diffuse, granular, or present beyond recognizable cells. | The expected IHC call requires identifiable neuronal cytoplasm (HPA tissue IHC: profile). Excess reagent, incomplete washing, or endogenous enzyme activity may create nonspecific background (general chromogenic IHC practice); appearance alone cannot identify which cause applies. | Inspect no-primary controls, review washing and blocking, and titrate the primary and detection reagents under the same staining conditions (general IHC practice). Score only cellular staining that remains distinguishable from background. |
| A ciliated-cell or Leydig-cell signal seems inconsistent with brain-enriched RNA. | HPA reports Medium staining in fallopian-tube ciliated cell bodies and testis Leydig cells while describing RNA as brain-enriched and staining–RNA agreement as low (HPA tissue IHC). Neither observation alone resolves antibody specificity. | Record the labeled cell type and compartment, compare the pattern with HPA's scored cells, and include specificity controls before treating the signal as confirmed NOS1AP (HPA tissue IHC; general IHC practice). |
| ICC-IF images show nucleoplasm or vesicles while the IHC slide shows neuronal cytoplasm. | HPA approves nucleoplasm and vesicles for ICC-IF and describes cytoplasmic neuronal tissue IHC staining (HPA subcellular; HPA tissue IHC). Those are observations from different assays and cellular settings. | Interpret each image against its own HPA reference and controls (HPA subcellular; HPA tissue IHC; general IF/IHC practice). Keep IF optimization on its separate guide page; this IHC decision concerns cellular staining in paraffin sections. |
Experiment evidence
IHC & IF images showing NOS1AP staining
A03060-3 · IHCIHC analysis of CAPON/NOS1AP using anti-CAPON/NOS1AP antibody (A03060-3). CAPON/NOS1AP was detected in a paraffin-embedded section of mouse
A03060-3 · IHCIHC analysis of CAPON/NOS1AP using anti-CAPON/NOS1AP antibody (A03060-3). CAPON/NOS1AP was detected in a paraffin-embedded section of rat br
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000198929-NOS1AP/tissue/cerebral+cortex#imid_18867403
HPA · HippocampusHuman Protein Atlas IHC, Hippocampus. Source: https://www.proteinatlas.org/ENSG00000198929-NOS1AP/tissue/hippocampal+formation#imid_18867404
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000198929-NOS1AP/tissue/colon#imid_18867337
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000198929-NOS1AP/tissue/liver#imid_18867322
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA030066; green = NOS1AP, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA030066; green = NOS1AP, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NOS1AP IHC & IF
🧪Run caudate first; neuronal cells should show medium staining (HPA: Caudate neuronal cells, Medium). Use adipose tissue adipocytes as the negative comparator (HPA: Adipocytes, Not detected); on the caudate slide, compare neuronal staining with adjacent cells lacking specific signal, without assuming every non-neuronal cell is NOS1AP-negative.
Positive control tissue: Caudate (Neuronal cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show NOS1AP in A-431, HEK293, U2OS, with annotated localisation: Nucleoplasm (approved), Vesicles (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a concentration-matched rabbit isotype control (hero caption: rabbit primary antibody; standard IHC practice). Use NOS1AP knockout tissue, if available, or peptide competition when the immunizing peptide is available; quench endogenous peroxidase before HRP/DAB detection, particularly around blood-containing areas (hero caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A03060-3 paraffin-section caption does not state the fixative (hero caption: fixative not stated). That caption used heat retrieval in EDTA at pH 8.0 for mouse brain, but does not establish that retrieval is required for every specimen (hero caption: mouse brain IHC). The supplied evidence does not establish whether frozen sections or IF are easier; brain autofluorescence should be checked with an unstained section if adapting this tissue to IF (HPA: Caudate neuronal staining, Medium; standard IF practice).
HPA tissue IHC evidence for NOS1AP
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low consistency between antibody staining and RNA expression data. Presumed off target binding observed and disregarded.). 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 | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | Medium | Protein (IHC) | HPA → |
| Fallopian tube | Ciliated cells (cell body) | Medium | Protein (IHC) | HPA → |
| Hippocampus | Neuronal cells | Medium | Protein (IHC) | HPA → |
| Testis | Leydig cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | Basal cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NOS1AP IHC Tips
Troubleshoot NOS1AP staining in paraffin sections by separating retrieval and detection problems from cell type, compartment, and antibody specificity.
How should I optimize retrieval when NOS1AP staining is weak?
Start with heat mediated retrieval in EDTA at pH 8.0 (datasheet A03060-3). The selected mouse brain paraffin section was stained after that retrieval, followed by 10% goat serum blocking and 2 μg/ml primary antibody overnight at 4°C (datasheet A03060-3). If staining is weak, vary heating time in small steps while holding section thickness, antibody concentration, and DAB development constant; inspect morphology after each change. Compare treated sections with a no primary control so stronger staining is not mistaken for retrieval dependent background. Record the heating method and cooling time, which the caption does not specify (datasheet A03060-3).
Could fixation explain variable NOS1AP staining across paraffin blocks?
Target specific fixation sensitivity is unknown: the selected paraffin section caption does not report a fixative (datasheet A03060-3). Record each block’s fixative, fixation duration, processing schedule, and storage history before comparing staining across batches. Keep retrieval at EDTA pH 8.0 and the primary antibody at 2 μg/ml initially, matching the documented staining conditions (datasheet A03060-3). If blocks differ, test matched sections under the same detection and DAB development conditions, then adjust one processing variable at a time. Do not interpret a faint block as NOS1AP negative until tissue preservation, counterstain, and an appropriate positive control have been assessed.
Which staining compartments are plausible for NOS1AP in tissue?
In tissue IHC, HPA reports cytoplasmic staining in neuronal cells, including medium staining in caudate, cerebral cortex, and hippocampus (HPA tissue IHC). UniProt places NOS1AP at filopodia and podosomes and reports no transmembrane segment (UniProt O75052 subcellular; UniProt O75052 topology). These annotations support checking cell bodies and projections where morphology allows, rather than requiring a sharp membrane outline. HPA also reports approved nucleoplasm and vesicle locations from ICC/IF, which need independent confirmation before assigning an IHC compartment (HPA subcellular). Score neuronal cytoplasm separately from nuclear, punctate, and diffuse staining, using adjacent morphology and a no primary control to assess unexpected patterns.
Can this antibody distinguish NOS1AP isoforms or phosphorylation states?
NOS1AP has 3 annotated isoforms and a PID domain spanning residues 26–196 (UniProt O75052 isoforms; UniProt O75052 domains). The supplied caption identifies the staining antibody and conditions but does not map its epitope or establish isoform selectivity (datasheet A03060-3). Phosphoserines are annotated at residues 188, 192, 195, 266, and 371; these annotations alone do not establish an effect on IHC binding (UniProt O75052 modified residues). Compare staining with an independently validated antibody to a defined region, or with genetic perturbation when available, before attributing regional differences to an isoform or phosphorylation state. Report the antibody identity and retrieval conditions alongside any such inference.
How can IF help verify a NOS1AP pattern seen by chromogenic IHC?
Use IF as an orthogonal localisation check and multiplex NOS1AP with a validated neuronal marker, because tissue IHC reports cytoplasmic expression in neuronal cells (HPA tissue IHC). Include single stain controls, then choose a far red NOS1AP channel if shorter wavelength tissue autofluorescence interferes with interpretation. NOS1AP has no transmembrane segment, while its mapped antibody epitope is not supplied; use controlled permeabilisation to test access to an intracellular epitope without assuming which side of a membrane it occupies (UniProt O75052 topology; datasheet A03060-3). HPA reports nucleoplasm and vesicles in ICC/IF, so inspect those compartments separately from neuronal cytoplasm (HPA subcellular; HPA tissue IHC). Do not transfer the paraffin section caption’s unreported fixation conditions to IF (datasheet A03060-3).
What should I change when NOS1AP DAB staining is diffuse?
First inspect a no primary control and compare DAB development time across sections; the selected protocol uses an HRP detection system with DAB (datasheet A03060-3). Quench endogenous peroxidase and check secondary antibody specificity as standard chromogenic IHC controls, particularly if staining appears outside recognizable cells. The documented primary concentration is 2 μg/ml with 10% goat serum blocking; titrate the primary around that starting point while keeping retrieval fixed at EDTA pH 8.0 (datasheet A03060-3). Shorter chromogen development or stronger washing may help if the no primary control is clean but background persists. Treat staining in HPA reported negative cell populations cautiously because presumed off target binding was noted (HPA tissue IHC).
How should I quantify NOS1AP staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the region and cell population before scoring; HPA reports medium neuronal staining in caudate, cerebral cortex, and hippocampus (HPA tissue IHC). For chromogenic sections, record the percentage of positive neuronal cells and an H-score from intensity categories, or count positive cells per mm² when cell density is the outcome. Normalize cell counts to the number of identifiable neuronal cells or measured tissue area, and apply identical counterstain, illumination, and DAB thresholds across batches. Exclude folds, section edges, and damaged tissue using predefined rules, then score blinded fields with the same sampling plan. Report retrieval at EDTA pH 8.0 and antibody concentration 2 μg/ml when using the documented conditions (datasheet A03060-3).
When is a NOS1AP positive signal likely to be artefactual?
A credible tissue result should fit the sampled cell population and compartment: HPA reports neuronal cytoplasmic staining, while UniProt also places NOS1AP at filopodia and podosomes (HPA tissue IHC; UniProt O75052 subcellular). Isolated staining at section edges, in necrotic areas, or in a no primary control warrants investigation before calling cells positive. Diffuse nuclear only staining needs separate validation because HPA tissue IHC emphasizes cytoplasm, although HPA ICC/IF reports nucleoplasm (HPA tissue IHC; HPA subcellular). Check endogenous peroxidase blocking when DAB deposits appear without primary antibody, and compare the pattern with an independent antibody or genetic control when feasible. Interpret discrepancies cautiously: HPA labels its tissue IHC approved but notes low RNA consistency and presumed off target binding (HPA tissue IHC).
Boster reagents
Best NOS1AP / Carboxyl-terminal PDZ ligand of neuronal nitric oxide synthase protein IHC Antibodies
Anti-NOS1AP A03060-3 has real IHC images from paraffin-embedded mouse, rat, and human brain sections (A03060-3 IHC image captions). No IF/ICC images are provided (A03060-3 IF image alts).
Real IHC data
Anti-CAPON/NOS1AP Antibody ®
Cat # A03060-3
The sole card is A03060-3, a rabbit antibody listed for IHC and human, mouse, and rat reactivity (A03060-3 catalog). Its IHC captions document staining in paraffin-embedded brain sections from all three species (A03060-3 IHC image captions).
Which to pick: For tissue IHC and cross-species studies, choose A03060-3: its own captions show paraffin-section staining in mouse, rat, and human brain using EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody (A03060-3 IHC image captions). The section fixative is unreported (A03060-3 IHC image captions). For IF/ICC, this payload offers no validated choice: A03060-3 has no IF/ICC application listing or IF images (A03060-3 catalog applications; IF image alts).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O75052 (CAPON_HUMAN, Carboxyl-terminal PDZ ligand of neuronal nitric oxide synthase protein).
- Human Protein Atlas. NOS1AP tissue IHC expression (reliability: Approved).
- Human Protein Atlas. NOS1AP subcellular location (ICC-IF): Localized to the nucleoplasm and vesicles..
- Human Protein Atlas. NOS1AP antibody validation summary (3 antibodies).
- A protein complex of SCRIB, NOS1AP and VANGL1 regulates cell polarity and migration, and is associated with breast cancer progression. Oncogene 2012 — PMC3419983.
- Inducible over-expression of cardiac Nos1ap causes short QT syndrome in transgenic mice. FEBS open bio 2023 — PMC9808597.
- PubMed PMID:9455484 — UniProt-cited evidence.
- PubMed PMID:16146415 — UniProt-cited evidence.
- PubMed PMID:16710414 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NOS1AP (Carboxyl-terminal PDZ ligand of neuronal nitric oxide synthase protein). Expected localisation Neuronal cytoplasm observed (HPA tissue IHC); filopodia and podosomes annotated (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A03060-3). Validated IHC antibody: A03060-3 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Caudate (Neuronal cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded mouse brain tissue; fixative not specified (datasheet A03060-3); 2. Fixation: Image fixative and duration unreported (datasheet A03060-3); 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: EDTA pH 8.0 (datasheet A03060-3); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A03060-3); 8. Primary antibody: Rabbit anti-NOS1AP, 2-5 μg/ml (datasheet A03060-3); 9. Primary incubation: Overnight at 4 °C (datasheet A03060-3). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Presumed off-target binding may confound scoring (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope coverage is unspecified (UniProt); Regulation: Expression varies by tissue (HPA tissue IHC). Evidence: UniProt O75052, HPA tissue IHC · Approved, HPA ICC-IF · nucleoplasm and vesicles., Datasheet A03060-3, PMC3419983. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NOS1AP/immunohistochemistry-ihc-guide.