This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
NCF1 / Neutrophil cytosol factor 1 · IHC design guide
Design Immunohistochemistry for NCF1
Use bone marrow hematopoietic cells or lymph node germinal center cells as high-staining IHC references (HPA tissue IHC). This guide helps assess staining in immune cell subsets (HPA tissue IHC) against the expected cytosolic and membrane distribution (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NCF1 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 | Cytosol and membrane expected (UniProt) | |
| Staining pattern | Immune cell subsets (HPA tissue IHC); cytosol or membrane expected (UniProt) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A01586-3) | |
| 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 | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A01586-3) | |
| Caveat | Endogenous peroxidase in phagocytes can mimic DAB signal (standard IHC practice) | |
| Regulation | Activation recruits NCF1 to membranes (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended NCF1 IHC & IF Protocols
The catalog antibody protocol is paired with published NCF1 IHC methods for rat submandibular gland (PMC13008658) and liver specimens (PMC9282929).
| Sample | Paraffin-embedded rat lymph tissue; fixative not specified (datasheet A01586-3) |
| Fixation | Image fixative and duration unreported (datasheet A01586-3); 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 A01586-3); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A01586-3) |
| Primary antibody | Rabbit anti-NCF1, 2-5 μg/ml (datasheet A01586-3) |
| Primary incubation | Overnight at 4 °C (datasheet A01586-3) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A01586-3) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | NCF1-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: Distinct expression in a subset of immune cells in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet A01586-3); adjust retrieval for the specimen if needed.
Section 2
What Is the Expected NCF1 Staining Pattern?
NCF1 is a cytosolic protein that can associate with membranes during phagocyte NADPH oxidase assembly; it has no transmembrane segment (UniProt P14598). In IHC, expect staining in subsets of immune cells across tissues, including strong staining in bone marrow hematopoietic cells and lymphoid populations, and moderate staining in lung macrophages (HPA tissue IHC). The tissue profile has Enhanced reliability, reflecting consistency between antibody staining and RNA expression (HPA tissue IHC).
| Cytoplasmic staining in scattered immune cells, with strong signal in bone marrow hematopoietic cells. | This matches the expected compartment (UniProt P14598) and a high-expression reference population (HPA: High in bone marrow hematopoietic cells). Score the stained cells rather than averaging signal across the entire section. |
| Staining is confined to nuclei, with little or no cytoplasmic signal. | An exclusively nuclear pattern conflicts with the reported cytosolic and membrane locations (UniProt P14598). Check the counterstain and detection controls before interpreting the nuclear signal as NCF1. |
| Strong signal fills adipocytes or respiratory epithelial cells. | These cells are reported as Not detected (HPA: adipose tissue adipocytes; bronchus respiratory epithelial cells). Consider nonspecific antibody binding or endogenous detection activity; nearby immune cells may still be positive (HPA tissue IHC). |
| Brown precipitate covers many cell types or extends beyond cell boundaries. | Diffuse chromogen obscures cell-specific scoring and may reflect background from detection or washing (general IHC practice). Compare a no-primary control and assess whether the signal follows identifiable cells. |
| No staining appears in bone marrow hematopoietic cells or lymph node germinal center cells. | Both are reported High (HPA tissue IHC). A blank section therefore needs a control and workflow check before being read as a biological negative; the HPA profile does not establish target-specific fixation sensitivity. |
💡Expected NCF1 appearanceCall positive when identifiable immune cells show predominantly cytoplasmic staining, potentially with peripheral association (UniProt P14598), strongest in the specified bone marrow and lymphoid populations and moderate in lung macrophages (HPA tissue IHC); uniform epithelial or exclusively nuclear signal is suspect (HPA tissue IHC; UniProt P14598).
| Cell population selected for scoring | NCF1 is detected in peripheral blood monocytes and neutrophils (UniProt P14598), while tissue staining is distinct in subsets of immune cells (HPA tissue IHC). Identify the cell population before calling a whole section positive or negative. |
| Cytosolic versus peripheral signal | NCF1 is cytosolic and participates in oxidase assembly at plasma or phagosomal membranes (UniProt P14598). Some peripheral staining can fit its biology; membrane association alone does not establish activation in a fixed section. |
| Antibody evidence | IHC validation differs among the listed antibodies: HPA047836 and CAB004524 are Enhanced; HPA052095 is Supported (HPA antibodies). This supports checking the exact antibody used when comparing staining patterns. |
| Isoform and epitope interpretation | Two isoforms are listed (UniProt P14598). The supplied record does not map antibody epitopes or establish isoform-specific IHC staining, so a negative result cannot identify which isoform is absent. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive tissue is blank. | The run may have failed, or the tested section may lack the expected cells; bone marrow hematopoietic cells are High (HPA tissue IHC). | Confirm the cells on a counterstained section, then review the IHC control, retrieval, antibody dilution and detection steps as general workflow checks. |
| Most of the section has diffuse brown background. | Residual detection reagent or inadequate washing can produce nonspecific chromogen deposition (general IHC practice). | Inspect a no-primary control and the pattern around cell boundaries; review blocking and washes before changing the biological interpretation. |
| Unexpected strong staining appears in epithelial cells or adipocytes. | Respiratory epithelial cells and adipocytes are Not detected in the specified HPA tissues (HPA tissue IHC); binding or detection activity may explain the signal. | Compare a no-primary control and cell morphology; score identifiable immune cells separately from the unexpected cell population. |
| Signal looks nuclear rather than cytoplasmic. | Nuclear-only staining does not match the reported cytosolic and membrane locations (UniProt P14598). | Check focus, counterstain and a no-primary control, then compare with the cytoplasmic pattern in a known-positive immune-cell population. |
| A thin peripheral rim is the only visible signal. | Membrane association is biologically plausible during oxidase assembly (UniProt P14598), but the rim alone does not establish NCF1 specificity or activation. | Check whether the rim belongs to identifiable immune cells and whether control staining supports a cell-associated pattern. |
| Can the IHC pattern be used as the expected IF/ICC pattern? | HPA lists no main ICC-IF location or cell-line images, and the listed antibodies have no ICC validation entry (HPA subcellular; HPA antibodies). | Use an independently validated IF/ICC control and assess cellular localisation there; do not infer an IF/ICC protocol or image pattern from these IHC data. |
Experiment evidence
IHC & IF images showing NCF1 staining
A01586-3 · IHCIHC analysis of P47 Phox/NCF1 using anti-P47 Phox/NCF1 antibody (A01586-3). P47 Phox/NCF1 was detected in a paraffin-embedded section of rat
A01586-3 · IHCIHC analysis of P47 Phox/NCF1 using anti-P47 Phox/NCF1 antibody (A01586-3). P47 Phox/NCF1 was detected in a paraffin-embedded section of mou
A01586-3 · IFIF analysis of Phox/NCF1 using anti-Phox/NCF1 antibody (A01586-3). Phox/NCF1 was detected in a paraffin-embedded section of mouse lymph node
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000158517-NCF1/tissue/cerebral+cortex#imid_1353980
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000158517-NCF1/tissue/colon#imid_1353906
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000158517-NCF1/tissue/liver#imid_1353907
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000158517-NCF1/tissue/kidney#imid_1353908
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NCF1 IHC & IF
🧪Run bone marrow first and expect staining in hematopoietic cells (HPA: High in bone-marrow hematopoietic cells). Use adipose tissue as a negative comparator because adipocytes are listed as not detected (HPA: Not detected in adipocytes); within the bone-marrow slide, morphologically distinct stromal cells should show only background chromogen, but they are not validated negatives (HPA: High designation applies to hematopoietic cells).
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 carries no ICC-IF cell line for NCF1; derive a cell-line control from the positive tissue's cell type (Hematopoietic cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control, a matched rabbit IgG isotype control, and NCF1-knockout tissue as a biological negative (caption: rabbit primary antibody; standard IHC controls). Quench endogenous peroxidase and inspect blood-rich bone marrow for residual background before scoring DAB staining (standard chromogenic IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A01586-3 paraffin-section caption does not state a fixative (caption: fixative unreported). Heat retrieval with EDTA at pH 8.0 is documented for rat lymph tissue, but retrieval dependency was not tested (caption: heat-mediated EDTA retrieval). Paraffin-section IHC has a documented example; the supplied evidence does not establish that frozen sections or IF are easier, and endogenous peroxidase can complicate interpretation in blood-rich bone marrow (caption: paraffin-section IHC; HPA subcellular: no ICC-IF images; standard IHC practice).
HPA tissue IHC evidence for NCF1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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 → |
| Lymph node | Germinal center cells | High | Protein (IHC) | HPA → |
| Spleen | Cells in white pulp | High | Protein (IHC) | HPA → |
| Tonsil | Non-germinal center cells | High | Protein (IHC) | HPA → |
| Lung | Macrophages | 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 NCF1 IHC Tips
Troubleshoot NCF1 staining in paraffin sections by checking retrieval, immune cell distribution, cytosolic localisation and chromogenic background against the available evidence.
How should I retrieve NCF1 in paraffin sections when staining is weak?
Start with heat mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A01586-3). The selected image used this retrieval before overnight incubation at 4°C with 2 μg/ml primary antibody, so reproduce those conditions when assessing a weak result (datasheet A01586-3). Record heating and cooling times, since inconsistent retrieval can change staining across a batch (standard IHC practice). If signal remains weak, compare a small retrieval series while keeping antibody concentration and detection constant (standard IHC practice). Judge improvement in immune cell rich regions, where NCF1 expression is expected, rather than by an increase in diffuse tissue colour (HPA: immune cell subset; standard IHC practice).
Could fixation explain weak or uneven NCF1 staining in my paraffin sections?
NCF1 specific fixation sensitivity is unknown from the supplied evidence: the selected paraffin section caption does not state a fixative (datasheet A01586-3). Record the fixative, fixation duration, tissue thickness and processing history for each specimen before comparing staining intensity (standard IHC practice). Use similarly processed sections when adjusting retrieval or primary antibody conditions, because those comparisons are harder to interpret across different processing histories (standard IHC practice). Keep a known positive immune cell rich section in each run to distinguish a specimen problem from a run problem (HPA: distinct expression in a subset of immune cells; standard IHC practice). Do not assign a fixation effect from expression patterns alone (standard IHC practice).
Should NCF1 stain the cytoplasm or outline cell membranes in IHC?
Expect predominantly cytoplasmic staining, with possible membrane associated signal, because NCF1 is annotated in the cytosol and at membranes (UniProt P14598: subcellular location). NCF1 has no transmembrane segment; its recruitment to plasma or phagosomal membranes accompanies assembly of the activated oxidase complex (UniProt P14598: topology and function). Interpret a crisp membrane pattern in its cellular context rather than requiring every positive cell to show one (UniProt P14598: activation mechanism; standard IHC practice). Compare the distribution with nearby immune cells: NCF1 is detected in blood monocytes and neutrophils, and HPA reports expression in immune cell subsets across tissues (UniProt P14598: tissue specificity; HPA: tissue IHC profile).
How do isoforms and modified regions affect interpretation of NCF1 IHC?
NCF1 has 2 annotated isoforms and a PX domain at residues 4–125 plus SH3 domains at 156–215 and 226–285 (UniProt P14598: isoforms and domains). Several annotated phosphoserines occur near the C terminus, including positions 303, 304, 320, 328 and 345 (UniProt P14598: modified residues). The supplied caption does not identify the antibody epitope, so these annotations alone cannot establish which isoforms or phosphorylation states its IHC detects (datasheet A01586-3: caption; UniProt P14598: annotations). If results vary across specimens, obtain the immunogen or epitope information and test specificity with an independent antibody or suitable negative control before assigning a molecular explanation (standard IHC practice).
How can I use IF to check which cells contain NCF1?
Use IF as a separate validation experiment and multiplex NCF1 with a marker for the immune cell population being assessed, such as a macrophage marker when examining macrophages (HPA: lung macrophages, Medium; standard IF practice). Select fluorophores after checking tissue autofluorescence in an unstained section, and use spectrally separated channels with single stain controls (standard IF practice). Include permeabilisation sufficient to expose a cytosolic epitope, because NCF1 is cytosolic and has no transmembrane segment (UniProt P14598: subcellular location and topology; standard IF practice). Its membrane association can change during oxidase assembly, so compare intracellular distribution under matched conditions (UniProt P14598: function; standard IF practice). The paraffin IHC caption supplies no IF fixation or permeabilisation conditions (datasheet A01586-3).
How do I reduce diffuse brown staining without losing genuine NCF1 signal?
First compare a no primary control and an appropriate positive section; diffuse colour that persists without primary antibody points toward detection background (standard IHC practice). The selected paraffin image used 10% goat serum block, a peroxidase conjugated secondary and DAB development (datasheet A01586-3). Apply a peroxidase block and check secondary antibody controls when endogenous enzyme activity or nonspecific secondary binding is suspected (standard chromogenic IHC practice). Then titrate primary antibody around the documented 2 μg/ml condition while holding retrieval and development time constant (datasheet A01586-3; standard IHC practice). Retain staining that resolves to plausible immune cells rather than accepting stronger diffuse tissue colour as improved detection (HPA: immune cell subset; standard IHC practice).
What should I quantify when NCF1 positive cells are scattered through tissue? ⚠ ANSWER MARKED FOR VERIFICATION
Define the region and cell population before scoring, since NCF1 staining is reported in a subset of immune cells across tissues (HPA: tissue IHC profile). For a defined population, report the percentage of positive cells and, when intensity is reproducible, an H score based on intensity categories (standard IHC quantification practice). For dispersed cells, count positive cells per mm² of viable tissue and also report their fraction among the relevant immune cells (standard IHC quantification practice). Normalize comparisons to the same tissue compartment or eligible cell denominator, using matched staining and image analysis settings (standard IHC quantification practice). Exclude folds, edges and necrotic areas from scored regions using rules set before review (standard IHC quantification practice).
How can I distinguish true NCF1 staining from artefact?
A credible pattern places cytosolic or membrane associated signal in plausible immune cells, consistent with NCF1 localisation and its reported immune cell distribution (UniProt P14598: subcellular location; HPA: tissue IHC profile). Strong colour confined to section edges, folds or necrotic regions should trigger review of section quality and a no primary control (standard IHC practice). Diffuse nuclear staining is outside the supplied NCF1 localisation annotation and needs independent confirmation before being called positive (UniProt P14598: cytosol and membrane; standard IHC practice). Because DAB detection uses peroxidase in the selected image, investigate persistent colour in a no primary control as possible endogenous enzyme or detection background (datasheet A01586-3; standard IHC practice). Compare suspected positives with an immune cell rich reference processed in the same run (HPA: tissue IHC profile; standard IHC practice).
Boster reagents
Best NCF1 / Neutrophil cytosol factor 1 IHC Antibodies
A01586-3 has IHC images from rat and mouse paraffin lymph tissue and IF images from mouse and rat paraffin lymph node tissue (catalog image captions). Human reactivity is listed without a tissue image (catalog: reactivity).
Real IHC data
Anti-p47 phox/NCF1 Antibody ®
Cat # A01586-3
A01586-3 has IHC images from rat and mouse paraffin lymph tissue and IF images from mouse and rat paraffin lymph node tissue (catalog image captions). A01586S370 lists IHC and IF applications and Human, Mouse, and Rat reactivity, but has no supplied IHC or IF image captions (catalog: applications, reactivity, image captions).
Which to pick: Choose A01586-3 for paraffin-section IHC: its rat and mouse lymph tissue captions document EDTA retrieval, goat-serum blocking, and DAB detection; the fixative is unreported (A01586-3 IHC image captions). For IF, A01586-3 has mouse and rat paraffin lymph node images; ICC is not listed, while A01586S370 lists IF without a supplied image (catalog: applications and IF image captions). For work across Human, Mouse, and Rat, both SKUs list reactivity in all three species, but the supplied tissue images cover only Mouse and Rat for A01586-3 (catalog: reactivity and image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P14598 (NCF1_HUMAN, Neutrophil cytosol factor 1).
- Human Protein Atlas. NCF1 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. NCF1 subcellular location (ICC-IF): Highest expression in THP-1: 12.1 nTPM.
- Human Protein Atlas. NCF1 antibody validation summary (3 antibodies).
- Integrating machine learning and immune infiltration analysis to identify core genes and construct a diagnostic model for type 2 diabetes mellitus. Frontiers in endocrinology 2026 — PMC13008658.
- Deficiency of the Purinergic Receptor 2X(7) Attenuates Nonalcoholic Steatohepatitis Induced by High-Fat Diet: Possible Role of the NLRP3 Inflammasome. Oxidative medicine and cellular longevity 2017 — PMC5705892.
- Deficient production of reactive oxygen species leads to severe chronic DSS-induced colitis in Ncf1/p47phox-mutant mice. PloS one 2014 — PMC4038546.
- Distinct histopathological phenotypes of severe alcoholic hepatitis suggest different mechanisms driving liver injury and failure. The Journal of clinical investigation 2022 — PMC9282929.
- PubMed PMID:2550933 — UniProt-cited evidence.
- PubMed PMID:2547247 — UniProt-cited evidence.
- PubMed PMID:2398896 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NCF1 (Neutrophil cytosol factor 1). Expected localisation Cytosol and membrane expected (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A01586-3). Validated IHC antibody: A01586-3 at 2-5 μg/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 rat lymph tissue; fixative not specified (datasheet A01586-3); 2. Fixation: Image fixative and duration unreported (datasheet A01586-3); 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 A01586-3); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A01586-3); 8. Primary antibody: Rabbit anti-NCF1, 2-5 μg/ml (datasheet A01586-3); 9. Primary incubation: Overnight at 4 °C (datasheet A01586-3). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A01586-3); Caveat: Endogenous peroxidase in phagocytes can mimic DAB signal (standard IHC practice); Isoform / epitope: 2 isoforms; epitope coverage is unspecified (UniProt); Regulation: Activation recruits NCF1 to membranes (UniProt). Evidence: UniProt P14598, HPA tissue IHC · Enhanced, HPA cell-line RNA · Highest expression in THP-1: 12.1 nTPM, Datasheet A01586-3, PMC13008658, PMC9282929. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NCF1/immunohistochemistry-ihc-guide.