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NCF2 / Neutrophil cytosol factor 2 · IHC design guide
Design Immunohistochemistry for NCF2
Plan NCF2 paraffin IHC around its expected cytoplasmic location and the observed enrichment in neutrophils and macrophages (UniProt; HPA tissue IHC). This guide covers tissue controls, antibody dilution, DAB detection, and cell-specific scoring (HPA tissue IHC; datasheet A02476-1; standard IHC practice).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NCF2 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 | Expected cytoplasm (UniProt); tissue compartment unreported (HPA tissue IHC) | |
| Staining pattern | High in neutrophils and macrophages; compartment unreported (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A02476-1) | |
| Positive control | Bone marrow+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 paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Neutrophil endogenous peroxidase can mimic DAB signal (HPA tissue IHC; standard IHC practice) | |
| Regulation | Expression regulation unreported (UniProt) | |
| Isoform / epitope | 4 isoforms; cytoplasmic target; epitope coverage unknown (UniProt) |
Section 1
Recommended NCF2 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet: A02476-1) with three published NCF2 IHC protocols (PMC12936047; PMC12684989; PMC10134316).
| Sample | Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A02476-1) |
| Fixation | Image fixative and duration unreported (datasheet A02476-1); 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 A02476-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A02476-1) |
| Primary antibody | Rabbit anti-NCF2, 0.5-1μg/ml (datasheet A02476-1) |
| Primary incubation | Overnight at 4 °C (datasheet A02476-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A02476-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | NCF2-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: High expression in neutrophils and macrophages. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet: A02476-1); citrate pH 6.0 is a published alternative (PMC12936047).
Section 2
What Is the Expected NCF2 Staining Pattern?
NCF2 is a cytoplasmic protein with no transmembrane segment (UniProt P19878: subcellular location and topology). In paraffin-section IHC, expect staining chiefly in neutrophils and macrophages (HPA: tissue IHC profile). HPA rates tissue staining reliability as Enhanced, with medium consistency between antibody staining and RNA expression; use cell identity and compartment alongside intensity when judging a result (HPA: reliability).
| Cytoplasmic staining in identifiable neutrophils or macrophages, with limited staining in neighboring cells. | This fits the expected cell distribution and compartment (HPA: high expression in neutrophils and macrophages; UniProt P19878: cytoplasm). Lung macrophages are recorded at Medium, so a valid positive need not be the darkest cell on the slide (HPA: lung macrophages, Medium). |
| Predominantly nuclear staining, or a sharp membrane outline with little cytoplasmic signal. | Reassess specificity and slide interpretation: HPA supports cytosolic ICC-IF localization, while UniProt assigns NCF2 to the cytoplasm and reports no transmembrane segment (HPA: subcellular; UniProt P19878: topology). Activated NCF2 can join an oxidase complex at the plasma or phagosomal membrane, so membrane-associated signal alone is not proof of artefact (UniProt P19878: function). |
| Strong staining in adipocytes or bronchial respiratory epithelial cells. | Those cell types are listed as Not detected in the sampled tissues (HPA: adipose adipocytes; bronchus respiratory epithelium). Check whether stained cells were misidentified, then consider antibody cross-reactivity or endogenous chromogen-generating activity; either possibility requires controls before assigning NCF2 (general IHC practice). |
| Uniform haze across unrelated cells, extracellular space, or the whole section. | A broad haze is difficult to reconcile with the cell-selective tissue profile (HPA: high expression in neutrophils and macrophages). Excess primary antibody, inadequate blocking or washing, and detection background are general IHC possibilities; the supplied HPA and UniProt records do not identify a target-specific cause. |
| No convincing signal in bone-marrow hematopoietic cells or spleen red-pulp cells. | These are reported High-staining populations (HPA: bone marrow hematopoietic cells; spleen red-pulp cells). First confirm that the relevant cells are present and recognizable, then investigate assay performance; one blank section does not overturn the HPA pattern or establish absent NCF2 expression. |
💡Expected NCF2 appearanceCall a result positive when identifiable neutrophils or macrophages show convincing cytoplasmic staining (HPA: tissue IHC profile; UniProt P19878: cytoplasm); expect a Medium lung-macrophage or High bone-marrow-cell reference signal (HPA: tissue IHC), and treat widespread unrelated staining as suspect (general IHC practice).
| Cell population and reference tissue | Bone-marrow hematopoietic cells and spleen red-pulp cells are High; lung macrophages and lymph-node non-germinal-center cells are Medium (HPA: tissue IHC). Compare the intended cells, since a whole-section intensity impression can obscure a localized positive population (general IHC practice). |
| Cytosolic location and activation | HPA supports cytosolic localization in ICC-IF, and UniProt lists cytoplasm with no transmembrane segment (HPA: subcellular; UniProt P19878: topology). Assembly with the membrane oxidase core can occur on activation (UniProt P19878: function), so interpret a changed distribution in its cell context. |
| Antibody evidence | Three listed antibodies have Enhanced IHC status (HPA: HPA002327, HPA006040, CAB022160). That supports the reported tissue pattern; it does not guarantee specificity for every staining run or convert HPA's medium RNA–staining consistency into perfect agreement (HPA: validation and reliability). |
| What should IF/ICC show? | A cytosolic signal is the supported localization (HPA: subcellular, ICC-IF). Treat this as an interpretation cross-check for the separate IF/ICC guide, rather than an IHC-P protocol or evidence that fluorescence and chromogenic staining will have identical intensity. |
| Situation | Likely cause | Next action |
|---|---|---|
| Bone-marrow or spleen reference section appears blank. | The expected High-staining population may be missing from the field, or the IHC workflow may have failed (HPA: bone marrow and spleen, High; general IHC practice). | Locate the reported cell population on the counterstain, then check primary-antibody use, detection reagents, and a concurrently processed positive control (general IHC practice). |
| Only nuclei stain strongly. | The result conflicts with the supported cytosolic assignment (HPA: subcellular; UniProt P19878: cytoplasm); nonspecific staining or misread counterstain is possible (general IHC practice). | Inspect stained-cell morphology and a primary-omission control; repeat with an independently validated antibody if available (HPA: multiple Enhanced IHC antibodies; general IHC practice). |
| Granular brown signal persists in a primary-omission control. | Endogenous enzyme activity or detection-reagent background can produce chromogenic signal without primary antibody (general IHC practice). | Review the detection chemistry and its endogenous-activity blocking step, then reassess the same cell population against the control (general IHC practice). |
| Signal spreads across nearly every cell or the section edge. | Excess reagent, inadequate washes, or edge-related staining are general IHC possibilities; they do not match HPA's cell-selective profile (HPA: tissue IHC; general IHC practice). | Compare an unaffected interior field and a primary-omission control; optimize blocking, washes, or primary concentration using the antibody's documented IHC conditions (general IHC practice). |
| Adipocytes or bronchial epithelial cells appear strongly positive. | Those listed cell types are Not detected by HPA tissue IHC; cell misidentification, cross-reactivity, or detection background may explain the discrepancy (HPA: adipose and bronchus; general IHC practice). | Verify cell identity on morphology and compare a primary-omission control; seek agreement with another Enhanced IHC antibody before calling the staining NCF2 (HPA: antibody validation; general IHC practice). |
| A macrophage-rich field stains less intensely than bone marrow. | Intensity differs among HPA reference populations: lung macrophages are Medium while bone-marrow hematopoietic cells are High (HPA: tissue IHC). | Score the relevant cells against their own tissue reference and background, rather than requiring bone-marrow-level intensity in lung macrophages (HPA: tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing NCF2 staining
A02476-1 · IHCIHC analysis of NOXA2/p67phox/NCF2 using anti-NOXA2/p67phox/NCF2 antibody (A02476-1). NOXA2/p67phox/NCF2 was detected in paraffin-embedded s
A02476-1 · IHCIHC analysis of NOXA2/p67phox/NCF2 using anti-NOXA2/p67phox/NCF2 antibody (A02476-1). NOXA2/p67phox/NCF2 was detected in paraffin-embedded s
M02476-1 · IHCFigure 2. IHC analysis of NOXA2/p67phox/NCF2 using anti-NOXA2/p67phox/NCF2 antibody (M02476-1). NOXA2/p67phox/NCF2 was detected in a paraffi
M02476-1 · IHCFigure 3. IHC analysis of NOXA2/p67phox/NCF2 using anti-NOXA2/p67phox/NCF2 antibody (M02476-1). NOXA2/p67phox/NCF2 was detected in a paraffi
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000116701-NCF2/tissue/cerebral+cortex#imid_6192728
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000116701-NCF2/tissue/colon#imid_6192646
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000116701-NCF2/tissue/liver#imid_6192655
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000116701-NCF2/tissue/kidney#imid_6192648
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA006040; green = NCF2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA006040; green = NCF2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NCF2 IHC & IF
🧪Run bone marrow first and score staining in hematopoietic cells (HPA: High in bone marrow hematopoietic cells). Use adipose tissue adipocytes as the negative comparator (HPA: Not detected in adipocytes); on the bone marrow slide, assess morphologically identified nonhematopoietic cells as an internal background reference without assuming they are NCF2-negative (standard IHC practice).
Positive control tissue: Bone marrow (Hematopoietic cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show NCF2 in A-431, U-251MG, U2OS, with annotated localisation: Cytosol (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; a concentration-matched rabbit IgG isotype control matched to the primary antibody’s clonality; and NCF2 knockout material or a cognate peptide-block control if available (selected-SKU caption: rabbit primary; standard IHC practice). Check endogenous peroxidase and biotin background in bone marrow, especially if using the caption’s biotin-based detection with DAB (selected-SKU caption: SABC and DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the fixative in the selected A02476-1 paraffin-section caption is unreported (selected-SKU caption). Heat retrieval in EDTA at pH 8.0 was used in that caption, but retrieval dependence is unestablished (selected-SKU caption). The supplied evidence does not establish whether frozen sections or IF are easier; for bone marrow IHC, assess endogenous peroxidase and biotin background during interpretation (HPA: High in bone marrow hematopoietic cells; standard IHC practice).
HPA tissue IHC evidence for NCF2
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 |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Endometrium | Endothelial cells | High | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | High | Protein (IHC) | HPA → |
| Lung | Macrophages | Medium | Protein (IHC) | HPA → |
| Lymph node | Non-germinal center 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 → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NCF2 IHC Tips
Use cell type, cytosolic localisation and matched controls to troubleshoot NCF2 staining in paraffin section IHC (HPA tissue IHC; HPA subcellular).
Which retrieval condition should I try first when NCF2 staining is weak?
Use heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin section IHC with A02476-1 (datasheet A02476-1). The selected tonsil image used that condition, followed by 1 μg/ml primary antibody overnight at 4°C (caption A02476-1). Compare a retrieved section with a matched section lacking retrieval, and keep primary incubation and DAB development consistent (standard IHC practice). Include a tonsil reference section so a weak test specimen can be distinguished from a failed staining run (caption A02476-1; standard IHC practice). If staining remains weak, vary retrieval time cautiously and inspect morphology before changing buffer or pH (standard IHC practice).
How should I troubleshoot fixation when paraffin section NCF2 staining varies?
Target-specific fixation sensitivity for NCF2 is unknown from the supplied evidence; the selected paraffin section caption does not state its fixative (caption A02476-1). Record fixative, fixation duration and processing history for each specimen before comparing staining intensity (standard IHC practice). Process a reference section alongside the test sections and hold EDTA retrieval at pH 8.0 constant while investigating variation (datasheet A02476-1; standard IHC practice). Check tissue preservation and nuclear detail before assigning faint cytoplasmic staining to antigen loss (standard IHC practice; UniProt P19878: cytoplasm). Treat differences associated with fixation history as experimental observations requiring matched controls, not as an established NCF2-specific effect (standard IHC practice).
What staining pattern is plausible for NCF2 in chromogenic tissue IHC?
Expect predominantly cytoplasmic signal in positive cells because NCF2 is annotated in the cytoplasm and the cytosol is its supported subcellular location (UniProt P19878: cytoplasm; HPA subcellular). Examine neutrophils and macrophages first, since their expression is high in the tissue profile (HPA tissue IHC: high expression in neutrophils and macrophages). NCF2 helps assemble the phagocyte oxidase at plasma or phagosomal membranes during activation, so a peripheral accent may warrant investigation in context (UniProt P19878: function). Do not score isolated nuclear or diffuse extracellular DAB as the expected distribution without additional validation (UniProt P19878: cytoplasm; standard IHC practice). Compare morphology and a primary-omission control on the same specimen (standard IHC practice).
Could isoforms or epitope accessibility explain discordant NCF2 staining?
NCF2 has 4 annotated isoforms, but the supplied antibody caption does not identify its epitope or establish which isoforms it detects (UniProt P19878: isoforms; caption A02476-1). The protein has SH3 domains at residues 240–299 and 457–516, plus a PB1 domain at 351–429 (UniProt P19878: domains). Phosphothreonine 233 and phosphoserine 399 are annotated, yet their effects on this antibody's tissue staining are unreported (UniProt P19878: modified residues; caption A02476-1). Compare matched sections under the documented retrieval and incubation conditions before attributing discordance to an isoform or modification (datasheet A02476-1; standard IHC practice). Request epitope mapping or orthogonal evidence if isoform-specific interpretation is essential (standard IHC practice).
How can IF help resolve ambiguous NCF2-positive cells in an IHC section?
Use IF on matched material as a separate localisation check, and multiplex NCF2 with a validated neutrophil or macrophage marker because those are expected positive cell types (HPA tissue IHC: high expression in neutrophils and macrophages; standard IF practice). Select spectrally separated fluorophores and compare unstained tissue in each channel to identify autofluorescence (standard IF practice). Because NCF2 is cytosolic and has no transmembrane segment, include permeabilisation when testing access to a cytosolic epitope (HPA subcellular: cytosol; UniProt P19878: topology; standard IF practice). Use single-stain and primary-omission controls to assess bleed-through and nonspecific fluorescence (standard IF practice). Interpret colocalisation by cell morphology as well as channel overlap (standard IF practice).
What should I check when DAB obscures NCF2-positive cells?
Start with primary-omission and detection-only controls to separate antibody-associated staining from endogenous enzyme activity and detection background (standard IHC practice). Include an endogenous peroxidase block before DAB development, then compare matched sections with the same development time (standard chromogenic IHC practice). The selected tonsil image used 10% goat serum block, 1 μg/ml primary antibody and a biotin-based detection system (caption A02476-1). If background persists, check the serum block, primary concentration and biotin-based detection controls one variable at a time (caption A02476-1; standard IHC practice). Evaluate staining against cell morphology because the HPA profile includes positive immune cell populations within tissue (HPA tissue IHC).
How should I quantify NCF2 IHC across specimens with different immune cell content? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring: NCF2 is cytosolic and the tissue profile highlights neutrophils and macrophages (HPA subcellular; HPA tissue IHC). Score the percentage of positive cells and staining intensity, or calculate an H-score within the same prespecified cell population (standard IHC scoring practice). For infiltration questions, report positive cell density per mm² of viable tissue and normalise positive cells to all cells of the corresponding identified type (standard histology practice). Keep section selection, counterstain and DAB development comparable across cases, and exclude folds and necrosis using consistent rules (standard IHC practice). Report cell-type composition alongside staining scores so shifts in immune cell abundance remain visible (standard IHC scoring practice).
How can I distinguish genuine NCF2 signal from artefact?
A credible positive has cytoplasmic staining in an identifiable cell; NCF2 is cytosolic and its tissue profile highlights neutrophils and macrophages (HPA subcellular; HPA tissue IHC). Check whether the pattern recurs in a reference section, such as tonsil processed with the documented EDTA pH 8.0 retrieval (caption A02476-1; standard IHC practice). Treat isolated nuclear deposits, section-edge staining and staining confined to necrotic material as suspect until matched controls resolve them (UniProt P19878: cytoplasm; standard IHC practice). Use primary-omission and peroxidase controls when deposits could reflect endogenous enzyme activity or detection background (standard chromogenic IHC practice). Interpret unexpected cell populations cautiously because the HPA staining profile has only medium consistency with RNA expression (HPA tissue IHC: Enhanced reliability description).
Boster reagents
Best NCF2 / Neutrophil cytosol factor 2 IHC Antibodies
Two anti-NCF2 antibodies have IHC images from human paraffin sections; one also has mouse and rat spleen images (catalog IHC captions). IF/ICC is listed for M02476-1 without an IF image (catalog applications and images).
Real IHC data
Anti-NOXA2/p67phox/NCF2 Antibody ®
Cat # A02476-1
Real IHC data
Anti-NOXA2/p67phox Rabbit Monoclonal Antibody
Cat # M02476-1
A02476-1 has IHC images from human tonsil and mouse and rat spleen paraffin sections (A02476-1 IHC captions). M02476-1 has IHC images from human spleen and tonsil paraffin sections and is listed for IF/ICC in human, mouse and rat (M02476-1 IHC captions; catalog applications and reactivity).
Which to pick: For tissue IHC, choose A02476-1 when images across human, mouse and rat matter, or M02476-1 when a rabbit monoclonal is preferred; both have paraffin section IHC images (catalog IHC captions; M02476-1 catalog host and clone). For IF/ICC, choose M02476-1 because IF/ICC is listed at 1:50, although no IF image is supplied (M02476-1 catalog applications, dilution and images). The IHC captions identify paraffin sections but do not report the fixative, so fixation conditions remain unreported for both SKUs (catalog IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P19878 (NCF2_HUMAN, Neutrophil cytosol factor 2).
- Human Protein Atlas. NCF2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. NCF2 subcellular location (ICC-IF): Localized to the cytosol..
- Human Protein Atlas. NCF2 antibody validation summary (3 antibodies).
- NCF2 facilitates M2 macrophage polarization in glioblastoma through activation of the notch1-osteopontin axis. Frontiers in immunology 2026 — PMC12936047.
- NCF2 as a Novel Diagnostic Biomarker in Ulcerative Colitis: Linking Macrophage Infiltration to Immune Dysregulation via Multi-Omics Analysis. Journal of inflammation research 2025 — PMC12684989.
- Novel Diagnostic Biomarkers Related to Oxidative Stress and Macrophage Ferroptosis in Atherosclerosis. Oxidative medicine and cellular longevity 2022 — PMC9410850.
- Role of NCF2 as a potential prognostic factor and immune infiltration indicator in hepatocellular carcinoma. Cancer medicine 2023 — PMC10134316.
- PubMed PMID:1692159 — UniProt-cited evidence.
- PubMed PMID:7903171 — UniProt-cited evidence.
- PubMed PMID:12207919 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NCF2 (Neutrophil cytosol factor 2). Expected localisation Expected cytoplasm (UniProt); tissue compartment unreported (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A02476-1). Validated IHC antibody: A02476-1 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Bone marrow (Hematopoietic cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A02476-1); 2. Fixation: Image fixative and duration unreported (datasheet A02476-1); 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 A02476-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A02476-1); 8. Primary antibody: Rabbit anti-NCF2, 0.5-1μg/ml (datasheet A02476-1); 9. Primary incubation: Overnight at 4 °C (datasheet A02476-1). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Neutrophil endogenous peroxidase can mimic DAB signal (HPA tissue IHC; standard IHC practice); Isoform / epitope: 4 isoforms; cytoplasmic target; epitope coverage unknown (UniProt); Regulation: Expression regulation unreported (UniProt). Evidence: UniProt P19878, HPA tissue IHC · Enhanced, HPA ICC-IF · cytosol., Datasheet A02476-1, PMC12936047, PMC12684989. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NCF2/immunohistochemistry-ihc-guide.