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NOXO1 / NADPH oxidase organizer 1 · IHC design guide
Design Immunohistochemistry for NOXO1
Plan NOXO1 chromogenic IHC in paraffin sections using duodenal endocrine cells and small-intestinal enterocytes as positive controls (HPA tissue IHC). This guide covers the catalog antibody’s documented IHC workflow (datasheet A05047-2), expected cytoplasmic staining (HPA tissue IHC), and isoform-aware interpretation (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NOXO1 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 | Predominantly cytoplasmic tissue staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in duodenal endocrine cells and enterocytes (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A05047-2) | |
| Positive control | Duodenum+3 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 paraffin sections (standard IHC practice; not target-specific) | |
| Caveat | Antibody staining has medium concordance with RNA (HPA tissue IHC) | |
| Regulation | Isoform 3 is mainly expressed in colon (UniProt) | |
| Isoform / epitope | Four isoforms; no transmembrane segment; epitope coverage unknown (UniProt) |
Section 1
Recommended NOXO1 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: A05047-2) is accompanied by one published NOXO1 chromogenic IHC protocol (PMC5951971).
| Sample | Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A05047-2) |
| Fixation | Image fixative and duration unreported (datasheet A05047-2); 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 A05047-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A05047-2) |
| Primary antibody | Rabbit anti-NOXO1, 2-5 μg/ml (datasheet A05047-2) |
| Primary incubation | Overnight at 4 °C (datasheet A05047-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A05047-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | NOXO1-positive staining in endocrine cells of duodenum (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression most abundant in gastrointestinal tract. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet: A05047-2). The published protocol specifies boiling in retrieval buffer but does not give its pH (PMC5951971).
Section 2
What Is the Expected NOXO1 Staining Pattern?
In paraffin section IHC, expect predominantly cytoplasmic NOXO1 staining in gastrointestinal cells, especially duodenal endocrine cells and small intestinal enterocytes (HPA: cytoplasmic expression most abundant in gastrointestinal tract; High in both cell types). High staining is also reported in elongated or late spermatids (HPA: High in testis). NOXO1 associates with the cell membrane but has no transmembrane segment (UniProt Q8NFA2: subcellular location and topology). HPA rates tissue IHC reliability Enhanced, with medium consistency between staining and RNA data (HPA: tissue IHC).
| Strong cytoplasmic staining in duodenal endocrine cells or small intestinal enterocytes. | This matches the reported positive cells and compartment (HPA: High in both cell types; cytoplasmic expression). Compare cells within the section; a brown deposit alone does not identify its source (standard IHC interpretation). |
| Cytoplasmic staining in elongated or late spermatids, with little staining in adjacent cells. | This fits the reported testis pattern (HPA: High in elongated or late spermatids). UniProt reports preferential expression of isoform 1 in testis; routine IHC cannot assign the stained isoform without antibody-specific evidence (UniProt Q8NFA2: isoforms and tissue specificity; standard IHC interpretation). |
| Predominantly nuclear staining in a section expected to show cytoplasmic signal. | Treat this as a suspect compartment pattern: HPA reports cytoplasmic tissue staining, while UniProt describes membrane association (HPA: tissue IHC; UniProt Q8NFA2: subcellular location). Review controls and staining distribution before calling it NOXO1 (standard IHC interpretation). |
| Strong staining in adipocytes or adrenal glandular cells, especially without the expected positive-cell pattern. | These cells were reported as not detected (HPA: adipose adipocytes and adrenal glandular cells). Consider cross-reactivity or endogenous detection activity; neither can be diagnosed from appearance alone (standard IHC interpretation). |
| Uniform color across cells, extracellular spaces, and section edges. | A distribution that ignores cell boundaries is more consistent with background than a cell-specific pattern (standard IHC interpretation). Compare a primary-antibody omission control and the expected positive cells before scoring (HPA: High in duodenal endocrine cells and small intestinal enterocytes; standard IHC practice). |
💡Expected NOXO1 appearanceCall a section positive when cytoplasmic staining is clearly enriched in duodenal endocrine cells, small intestinal enterocytes, or elongated or late spermatids (HPA: High in each); widespread nuclear or cell-independent color is suspect background (HPA: cytoplasmic profile; standard IHC interpretation).
| Compartment and topology | HPA describes cytoplasmic tissue staining; UniProt places NOXO1 at the cell membrane and reports no transmembrane segment (HPA: tissue IHC; UniProt Q8NFA2: subcellular location and topology). A membrane-adjacent component can be considered alongside cytoplasmic staining, but a crisp membrane rim is not required by the supplied IHC evidence. |
| Cell type and tissue selection | Duodenal endocrine cells, small intestinal enterocytes, and elongated or late spermatids provide reported high-staining comparators; salivary glandular cells are medium (HPA: tissue IHC). Adipocytes and adrenal glandular cells were not detected, so their staining needs scrutiny (HPA: tissue IHC). |
| Isoform distribution | UniProt lists four isoforms: isoform 3 mainly in colon and isoform 1 preferentially in testis (UniProt Q8NFA2: isoforms and tissue specificity). Those expression notes do not establish which isoforms an IHC antibody recognizes or a colon cell staining pattern (standard IHC interpretation). |
| Strength of tissue evidence | The reported tissue pattern carries an Enhanced IHC rating for HPA071540, while HPA also notes medium consistency with RNA expression (HPA: antibody validation and tissue IHC). Interpret an individual section against its cell-specific pattern and controls; the rating does not validate every compartment or IF result (standard IHC interpretation). |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in duodenal endocrine cells or small intestinal enterocytes. | A technical failure is possible because these are reported high-staining cells; a negative slide alone cannot distinguish failed detection from specimen variation (HPA: High in both; standard IHC interpretation). | Check the IHC-validated antibody's stated dilution and retrieval instructions, reagent activity, and an independently stained positive section; do not infer NOXO1-specific fixation sensitivity (standard IHC practice). |
| Weak or patchy signal in a reported positive tissue. | The stained cell population may be sparse or unevenly sampled; HPA's High level refers to specified cell types, not every cell in the organ (HPA: tissue IHC; standard IHC interpretation). | Find the relevant cells on the counterstained section, then compare replicate sections and the positive control under the same scoring conditions (standard IHC practice). |
| Diffuse brown background obscures cell boundaries. | Nonspecific reagent binding or endogenous chromogenic activity may contribute (standard IHC practice). The appearance does not by itself establish NOXO1 expression. | Inspect primary-antibody omission and detection-only controls; review blocking, washes, and endogenous enzyme quenching as applicable to the detection system (standard IHC practice). |
| Adipocytes or adrenal glandular cells stain strongly. | This conflicts with the reported not-detected pattern and raises possible cross-reactivity or endogenous detection activity (HPA: adipocytes and adrenal glandular cells not detected; standard IHC interpretation). | Compare omission controls and a reported high-staining cell type processed in the same run before assigning a positive score (HPA: tissue IHC; standard IHC practice). |
| Staining is predominantly nuclear despite a clear positive control. | The compartment differs from HPA's cytoplasmic tissue profile and UniProt's membrane association; identity remains uncertain (HPA: tissue IHC; UniProt Q8NFA2: subcellular location). | Check the counterstain and detection controls, then review the cell-specific distribution. Record the compartment discrepancy rather than scoring nuclear staining as the expected pattern (standard IHC interpretation). |
| Q: Can this IHC pattern serve as an IF/ICC positive-control pattern? | HPA supplies no main IF/ICC location or cell lines with ICC-IF images for NOXO1; the listed HPA071540 validation is IHC Enhanced with no ICC status (HPA: subcellular and antibody records). | A: Treat IF/ICC localization as unverified here and use the separate IF/ICC guide for that application; establish its own controls before interpreting fluorescence (HPA: subcellular and antibody records; standard IF practice). |
Experiment evidence
IHC & IF images showing NOXO1 staining
A05047-2 · IHCIHC analysis of NOXO1 using anti-NOXO1 antibody (A05047-2). NOXO1 was detected in a paraffin-embedded section of human colon cancer tissue.
A05047-2 · IHCIHC analysis of NOXO1 using anti-NOXO1 antibody (A05047-2). NOXO1 was detected in a paraffin-embedded section of human prostate cancer tissu
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000196408-NOXO1/tissue/cerebral+cortex#imid_19122315
HPA · DuodenumHuman Protein Atlas IHC, Duodenum. Source: https://www.proteinatlas.org/ENSG00000196408-NOXO1/tissue/duodenum#imid_19123600
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000196408-NOXO1/tissue/colon#imid_19123609
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000196408-NOXO1/tissue/liver#imid_19123602
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NOXO1 IHC & IF
🧪Run small intestine first and require staining in enterocytes (HPA: High in small-intestine enterocytes). Use adipose tissue adipocytes as the negative comparison (HPA: Not detected in adipocytes); on the positive slide, neighboring cells without enterocyte staining should show only background (IHC control practice).
Positive control tissue: Duodenum (Endocrine cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for NOXO1; derive a cell-line control from the positive tissue's cell type (Endocrine cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control, a concentration-matched rabbit IgG isotype control, and a NOXO1-knockout sample if available (selected IHC caption: rabbit primary; standard IHC control practice). Quench endogenous peroxidase and check for background in the small-intestine section before interpreting DAB staining (standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A05047-2 paraffin-section caption does not report a fixative (selected IHC caption). The caption uses heat-mediated EDTA retrieval at pH 8.0, but does not establish whether retrieval is required (selected IHC caption). Are frozen sections or IF/ICC easier? The supplied evidence does not establish either; check small-intestine sections for luminal background in IHC and autofluorescence if evaluating IF (HPA: High in small-intestine enterocytes; standard IHC/IF practice).
HPA tissue IHC evidence for NOXO1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium 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 |
|---|---|---|---|---|
| Duodenum | Endocrine cells | High | Protein (IHC) | HPA → |
| Small intestine | Enterocytes | High | Protein (IHC) | HPA → |
| Testis | Elongated or late spermatids | High | Protein (IHC) | HPA → |
| Salivary gland | Glandular 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 | Endocrine cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NOXO1 IHC Tips
Troubleshoot NOXO1 staining in paraffin section IHC using the catalog antibody’s colon cancer example, tissue expression evidence, and appropriate controls.
Which antigen retrieval conditions should I start with for NOXO1 paraffin sections?
Start with heat mediated retrieval in EDTA at pH 8.0 for NOXO1 paraffin sections (datasheet A05047-2). The catalog antibody’s colon cancer example then used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and DAB detection (caption A05047-2). If staining is weak, compare retrieval duration on adjacent sections while holding antibody concentration and detection conditions constant; document tissue damage alongside signal (standard IHC practice). Include a section processed without primary antibody and a known positive cell population, such as small intestinal enterocytes, so stronger brown staining is not mistaken for improved specificity (HPA: High in enterocytes; standard IHC practice).
Could fixation explain weak or uneven NOXO1 staining?
NOXO1 specific fixation sensitivity is unknown because the selected paraffin section caption does not state a fixative (caption A05047-2). Record the fixative, fixation duration, processing history, and section age for each specimen before attributing differences to antigen abundance (standard IHC practice). Compare adjacent sections from the same block using the stated EDTA pH 8.0 retrieval and 2 μg/ml primary incubation overnight at 4°C, changing one processing variable at a time (datasheet A05047-2; standard IHC practice). Check morphology and a no primary control alongside staining, since uneven preservation or detection background can otherwise imitate a biological gradient (standard IHC practice).
Should NOXO1 appear at the membrane or in the cytoplasm?
Interpret NOXO1 as potentially membrane associated while assessing cytoplasmic staining in tissue: UniProt lists cell membrane localisation, whereas HPA reports predominantly cytoplasmic IHC expression in the gastrointestinal tract (UniProt Q8NFA2; HPA: tissue profile). NOXO1 has no transmembrane segment, and isoform 3 associates with the plasma membrane in a lipid dependent manner (UniProt Q8NFA2). Score epithelial cell compartments separately on the DAB section, using the same retrieval and detection settings across samples (standard IHC practice). Diffuse extracellular deposits or staining confined to cut edges should prompt review of background and morphology before any compartment assignment (standard IHC practice).
Can this antibody distinguish NOXO1 isoforms or reveal an epitope problem?
Do not assign an isoform from a positive DAB section unless the antibody’s mapped epitope and isoform coverage are known (standard IHC practice). NOXO1 has 4 isoforms; isoform 3 is mainly expressed in colon, while isoform 1 is preferentially expressed in testis (UniProt Q8NFA2). Its PX domain spans residues 1–131, and its two SH3 domains span 163–225 and 237–296; these boundaries do not identify this antibody’s epitope (UniProt Q8NFA2; caption A05047-2). Compare staining across documented positive tissues and use an independently characterised epitope reagent if isoform assignment matters, recording retrieval conditions for each antibody (HPA: tissue profile; standard IHC practice).
How should I assess NOXO1 by multiplex IF alongside chromogenic IHC?
Treat IF as a separate assay and validate its staining before comparing it with DAB intensity from the paraffin section example (caption A05047-2; standard IF practice). Pair NOXO1 with a marker identifying the expected cell population, such as enterocytes in small intestine, and inspect single stain controls for spectral bleed through (HPA: High in enterocytes; standard IF practice). Choose fluorophores after checking tissue autofluorescence in an unstained section, favouring channels with clear separation from that background (standard IF practice). Because NOXO1 lacks a transmembrane segment and can associate with the plasma membrane, establish whether the antibody recognises an accessible or intracellular epitope before selecting permeabilisation conditions (UniProt Q8NFA2; standard IF practice).
How can I reduce diffuse brown background without losing NOXO1 signal?
Inspect a no primary section and the tissue morphology before increasing washes or reducing primary antibody exposure (standard IHC practice). The catalog example used 10% goat serum, 2 μg/ml rabbit primary overnight at 4°C, a peroxidase conjugated secondary for 30 minutes at 37°C, and DAB (caption A05047-2). Use a peroxidase block and limit DAB development to control endogenous enzyme signal and overdevelopment; these are general chromogenic IHC measures, not NOXO1 specific validation (standard IHC practice). Compare background in unstained or no primary controls with expected cell restricted staining, and reject edge staining or necrotic deposits as evidence of NOXO1 (standard IHC practice).
What should I measure when comparing NOXO1 IHC across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the compartment and cell population before scoring, since HPA describes cytoplasmic gastrointestinal staining while UniProt lists cell membrane localisation (HPA: tissue profile; UniProt Q8NFA2). Use an H-score based on staining intensity and percentage of positive cells, or report percentage positive cells within a predefined epithelial region (standard IHC practice). Normalise counts to the number of evaluable cells or measured tissue area in mm², excluding folds, necrosis, and cut edges by the same rule in every section (standard IHC practice). Keep retrieval, the 2 μg/ml primary condition, DAB development, and imaging settings consistent, and report background corrected scores alongside controls (caption A05047-2; standard IHC practice).
What makes a NOXO1 positive IHC result convincing?
A convincing result has reproducible, cell associated staining in the expected compartment and population, with little signal in the no primary control (UniProt Q8NFA2; HPA: tissue profile; standard IHC practice). Compare high reported populations, including small intestinal enterocytes and duodenal endocrine cells, with a low or undetected reference population processed alongside them (HPA: High in enterocytes and duodenal endocrine cells; HPA: Not detected in adipocytes). Interpret membrane adjacent or cytoplasmic staining in context because NOXO1 lacks a transmembrane segment and HPA reports cytoplasmic gastrointestinal staining (UniProt Q8NFA2; HPA: tissue profile). Discount isolated edge staining, necrotic deposits, and persistent brown signal after omitting primary antibody as likely artefacts requiring further controls (standard IHC practice).
Boster reagents
Best NOXO1 / NADPH oxidase organizer 1 IHC Antibodies
A05047-2 has real IHC data from paraffin-embedded human colon and prostate cancer sections (catalog IHC captions). Human, mouse, and rat reactivity is listed (catalog reactivity).
Real IHC data
Anti-NOXO1 Antibody ®
Cat # A05047-2
A05047-2 was demonstrated by chromogenic IHC in paraffin-embedded human colon cancer tissue (catalog IHC caption). The same SKU was demonstrated in paraffin-embedded human prostate cancer tissue (catalog IHC caption).
Which to pick: Choose A05047-2 for paraffin-section IHC; its captions report EDTA retrieval and DAB detection, but do not report the fixative (catalog IHC captions). No IF/ICC application or image is listed for A05047-2, so IF/ICC suitability is unestablished (catalog applications and images). A05047-2 is polyclonal and lists human, mouse, and rat reactivity, but its documented IHC examples are human only (catalog dilution record, reactivity, and IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8NFA2 (NOXO1_HUMAN, NADPH oxidase organizer 1).
- Human Protein Atlas. NOXO1 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. NOXO1 subcellular location (ICC-IF): Highest expression in OE19: 5.1 nTPM.
- Human Protein Atlas. NOXO1 antibody validation summary (1 antibodies).
- NF-κB-induced NOX1 activation promotes gastric tumorigenesis through the expansion of SOX2-positive epithelial cells. Oncogene 2019 — PMC6756228.
- NoxO1 Controls Proliferation of Colon Epithelial Cells. Frontiers in immunology 2018 — PMC5951971.
- PubMed PMID:12473664 — UniProt-cited evidence.
- PubMed PMID:12657628 — UniProt-cited evidence.
- PubMed PMID:12716910 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NOXO1 (NADPH oxidase organizer 1). Expected localisation Predominantly cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A05047-2). Validated IHC antibody: A05047-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Duodenum (Endocrine cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A05047-2); 2. Fixation: Image fixative and duration unreported (datasheet A05047-2); 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 A05047-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A05047-2); 8. Primary antibody: Rabbit anti-NOXO1, 2-5 μg/ml (datasheet A05047-2); 9. Primary incubation: Overnight at 4 °C (datasheet A05047-2). Watch for: Fixation: Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific); Caveat: Antibody staining has medium concordance with RNA (HPA tissue IHC); Isoform / epitope: Four isoforms; no transmembrane segment; epitope coverage unknown (UniProt); Regulation: Isoform 3 is mainly expressed in colon (UniProt). Evidence: UniProt Q8NFA2, HPA tissue IHC · Enhanced, HPA cell-line RNA · Highest expression in OE19: 5.1 nTPM, Datasheet A05047-2, PMC5951971. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NOXO1/immunohistochemistry-ihc-guide.