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NFIA / Nuclear factor 1 A-type · IHC design guide
Design Immunohistochemistry for NFIA
Plan paraffin-section NFIA IHC around the nuclear staining reported across tissues (HPA tissue IHC). Use the catalog antibody’s documented IHC conditions (datasheet M03531) and assess signal in nuclei.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NFIA Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Nuclear staining across tissues (HPA tissue IHC) | |
| Staining pattern | Nuclear staining across tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M03531) | |
| Positive control | Adipose tissue+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | No specific expression regulator annotated (UniProt) | |
| Isoform / epitope | Four isoforms; epitope effects are unknown (UniProt) |
Section 1
Recommended NFIA IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by one published renal cell carcinoma TMA protocol (PMC10240150).
| Sample | Paraffin-embedded human intestinal cancer tissue; fixative not specified (datasheet M03531) |
| Fixation | Image fixative and duration unreported (datasheet M03531); 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 M03531); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M03531) |
| Primary antibody | Mouse monoclonal (clone 16H11) anti-NFIA, 0.5-1μg/ml (datasheet M03531) |
| Primary incubation | Overnight at 4 °C (datasheet M03531) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet M03531) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | NFIA-positive staining in adipocytes of adipose tissue (HPA tissue IHC: High). HPA tissue profile: Nuclear expression in all tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval (datasheet M03531); the published TMA method does not specify a retrieval step (PMC10240150).
Section 2
What Is the Expected NFIA Staining Pattern?
NFIA is a nuclear DNA-binding protein with no transmembrane segment (UniProt Q12857). In tissue IHC, expect nuclear staining across tissues, including HPA-reported high staining in adipocytes, glial cells, respiratory epithelial cells, and hematopoietic cells (HPA tissue IHC). HPA rates the tissue profile Enhanced, with medium consistency between antibody staining and RNA expression data (HPA tissue IHC).
| Distinct nuclear staining in adipocytes or glial cells, with nuclei identifiable by counterstain. | This matches NFIA’s nuclear localization (UniProt Q12857) and HPA’s high staining in adipocytes and glial cells (HPA tissue IHC). Compare staining among cells in the same section; HPA levels do not define a universal intensity cutoff. |
| Strong cytoplasmic staining with little or no nuclear staining in an otherwise interpretable section. | A cytoplasmic-only IHC pattern conflicts with the nuclear tissue profile (HPA tissue IHC; UniProt Q12857). HPA also lists cytosol as an additional ICC-IF location (HPA subcellular), so cytoplasmic signal alone is a reason to investigate, not proof of artefact. |
| Staining appears in unexpected cells while nearby HPA-reported high cells lack nuclear signal. | Check cell identity and the distribution of stain before calling this NFIA. HPA reports nuclear expression across tissues and supplies no negative cell population (HPA tissue IHC); cross-reactivity or endogenous detection activity are possibilities to test (general IHC practice). |
| Diffuse color covers nuclei, cytoplasm, and extracellular areas without clear cell boundaries. | This does not resolve the expected nuclear pattern (UniProt Q12857; HPA tissue IHC). Section-wide background can arise from nonspecific antibody binding or detection chemistry; controls and staining distribution help distinguish them (general IHC practice). |
| No nuclear staining appears in an HPA-reported high-staining tissue. | Adipose adipocytes, caudate or cortical glial cells, and bronchial respiratory epithelial cells are reported high examples (HPA tissue IHC). A blank result warrants a technical check, but HPA’s Enhanced rating and medium RNA agreement do not guarantee positivity in every specimen (HPA tissue IHC). |
💡Expected NFIA appearanceCall a result positive when discernible nuclear stain occurs in the relevant cells, especially HPA-reported high populations (HPA tissue IHC; UniProt Q12857); isolated diffuse cytoplasmic or extracellular color without convincing nuclear staining is suspect (general IHC practice).
| Localization and cell selection | NFIA is nuclear and binds DNA as a homodimer (UniProt Q12857). HPA reports nuclear expression in all tissues but names specific high-staining cell populations (HPA tissue IHC); score the identified cells and their nuclei, rather than treating every stained area as equivalent. |
| Strength of tissue evidence | The HPA tissue profile is Enhanced, with medium staining-to-RNA consistency, and lists no negative or low tissues (HPA tissue IHC). Use its high examples as comparison material; do not infer a validated negative tissue or a fixed positive-cell threshold. |
| Antibody validation | HPA006111 and HPA008884 are rabbit polyclonal antibodies rated Enhanced for IHC and ICC (HPA antibodies). That supports the reported pattern but does not establish performance for a different antibody or a particular antigen-retrieval setting (HPA antibodies; general IHC practice). |
| Isoforms and processing | UniProt lists four NFIA isoforms, a 1–509 chain, no signal peptide, and no transmembrane segment (UniProt Q12857). Those facts support interpreting a nuclear pattern; they do not identify which isoforms an antibody detects or predict section staining intensity. |
| Antigen retrieval | If signal is weak, review the catalog antibody’s IHC-P instructions and verify the chosen retrieval conditions with appropriate controls (general IHC practice). Neither HPA nor the supplied UniProt record reports NFIA-specific fixation sensitivity or an optimal retrieval condition. |
| IF/ICC Q&A: where should fluorescence appear? | Mainly in the nucleoplasm; HPA also lists cytosol as an additional location and rates the ICC evidence Enhanced (HPA subcellular; HPA antibodies). Interpret that observation on the separate IF/ICC guide page; it does not specify an IHC-P protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| An HPA-reported high tissue shows no nuclear stain. | Possible staining-run failure, incompatible antibody conditions, or a sample-specific difference (general IHC practice); HPA does not guarantee every section is positive (HPA tissue IHC). | Confirm tissue and cell identity, inspect controls, then check the catalog antibody’s IHC-P instructions and retrieval conditions (general IHC practice). Repeat before concluding that the tissue lacks NFIA. |
| Only cytoplasm is strongly colored. | The pattern differs from nuclear tissue IHC (HPA tissue IHC; UniProt Q12857), although cytosol is listed as an additional ICC-IF location (HPA subcellular). | Compare nuclear counterstain and matched controls; check antibody and detection background (general IHC practice). Treat cytoplasmic-only IHC as unresolved until nuclear staining can be assessed. |
| Unexpected cells stain while expected cells stay blank. | Cell misidentification, cross-reactivity, or endogenous detection activity may explain the mismatch (general IHC practice). HPA provides no certified negative tissue or cell population (HPA tissue IHC). | Recheck morphology and use antibody-omission and detection controls as appropriate (general IHC practice). Compare with an HPA-reported high cell population in the same or a separate section (HPA tissue IHC). |
| Brown color is diffuse throughout the section. | Nonspecific binding or endogenous chromogenic detection activity can obscure nuclear localization (general IHC practice). Diffuse extracellular color is not the HPA nuclear pattern (HPA tissue IHC). | Inspect omission and reagent controls; review blocking, washes, antibody concentration, and detection settings (general IHC practice). Reassess only where individual nuclei and cells remain distinguishable. |
| Nuclear signal is weak but background is low. | Limited assay sensitivity or the chosen staining conditions may contribute (general IHC practice). HPA’s qualitative High label does not prescribe an intensity threshold for a new run (HPA tissue IHC). | Verify positive controls and the catalog antibody’s IHC-P instructions; adjust one staining variable at a time and retain counterstain for nuclear assessment (general IHC practice). |
| A section shows patchy nuclear staining. | A staining-run or section-quality issue may produce uneven results (general IHC practice); HPA reports nuclear expression broadly but does not define uniform intensity in every cell (HPA tissue IHC). | Check whether staining follows cell identity or a section-wide gradient, and compare replicate or control sections before scoring the pattern (general IHC practice). |
Experiment evidence
IHC & IF images showing NFIA staining
M03531 · IHCIHC analysis of NFIA using anti-NFIA antibody (M03531). NFIA was detected in paraffin-embedded section of human intestinal cancer tissue. He
M03531 · IHCIHC analysis of NFIA using anti-NFIA antibody (M03531). NFIA was detected in paraffin-embedded section of human intestinal cancer tissue. He
M03531 · IFIF analysis of NFIA using anti-NFIA antibody (M03531). NFIA was detected in immunocytochemical section of A431 cells. Enzyme antigen retriev
PB10080 · IHCIHC analysis of NFIA using anti-NFIA antibody (PB10080). NFIA was detected in a paraffin-embedded section of mouse brain tissue. Heat mediat
PB10080 · IHCIHC analysis of NFIA using anti-NFIA antibody (PB10080). NFIA was detected in a paraffin-embedded section of rat brain tissue. Heat mediated
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000162599-NFIA/tissue/cerebral+cortex#imid_1953716
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000162599-NFIA/tissue/colon#imid_1953642
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000162599-NFIA/tissue/liver#imid_1953635
HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000162599-NFIA/tissue/pancreas#imid_1953594
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA006111; green = NFIA, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA006111; green = NFIA, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NFIA IHC & IF
🧪Run adipose tissue first and assess nuclear staining in adipocytes (HPA: High in adipocytes; UniProt Q12857: nucleus). HPA detects NFIA in all 45 scored tissues, so there is no HPA-supported negative tissue; use no-primary and isotype controls for the negative comparison, and treat any unstained cells on the positive slide only as local background comparators, not validated NFIA-negative cells (HPA: no negative rows; NFIA detected in all 45 scored tissues).
Positive control tissue: Adipose tissue (Adipocytes, HPA High)
Negative control tissue: None in HPA: NFIA is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show NFIA in A-431, U-251MG, U2OS, NIH 3T3, with annotated localisation: Nucleoplasm (enhanced) (HPA subcellular).
Technical controls: Run a no-primary, secondary-only section and a concentration-matched mouse IgG isotype control alongside the catalog antibody, then confirm specificity with NFIA knockout material processed in the same way where available (selected M03531 caption: mouse primary antibody; standard IHC control practice). Block endogenous peroxidase and check for endogenous biotin background because the reported detection uses a biotinylated secondary, streptavidin–biotin complex and DAB (selected M03531 caption; standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected M03531 paraffin-section caption does not state the fixative (selected M03531 caption). That caption uses heat retrieval in EDTA at pH 8.0, but does not establish whether retrieval is required; the supplied evidence also does not establish that frozen sections or IF are easier (selected M03531 caption; HPA: ICC-IF images listed without a matched method comparison). In adipose tissue, lipid-rich areas can make section integrity and nuclear scoring difficult, so assess intact adipocyte nuclei (HPA: High in adipocytes; standard IHC practice).
HPA tissue IHC evidence for NFIA
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 |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Adipocytes | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: NFIA is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced NFIA IHC Tips
These questions address nuclear NFIA staining in paraffin section chromogenic IHC, with one entry on interpreting complementary IF experiments.
Which retrieval condition should I start with when nuclear NFIA staining is weak?
Start with heat mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet M03531). The selected image used this condition before incubation with 1 µg/ml antibody overnight at 4°C; its caption does not report the fixative (datasheet M03531). If nuclear staining remains weak, compare retrieval duration on adjacent sections while keeping antibody concentration and detection constant (standard IHC practice). Check tissue integrity and background after heating, because excessive retrieval can make a stronger signal harder to interpret (standard IHC practice). Score nuclei against a matched section processed without primary antibody (standard IHC practice).
How should I troubleshoot weak NFIA staining when fixation details are uncertain?
The selected paraffin section caption does not state its fixative, so NFIA sensitivity to any specific fixation condition is unknown (datasheet M03531). Record the available fixation and processing history for each specimen, then compare sections with similar histories before changing retrieval or antibody concentration (standard IHC practice). If a known positive section stains while the test section does not, inspect preservation and repeat EDTA retrieval at pH 8.0 on an adjacent section (datasheet M03531; standard IHC practice). Do not infer fixation sensitivity from nuclear localisation, phosphorylation sites, or tissue staining patterns (UniProt Q12857; HPA tissue IHC).
What staining pattern should count as convincing NFIA in paraffin sections?
Prioritise a nuclear DAB signal in morphologically intact cells: NFIA is annotated as nuclear, and tissue IHC reports nuclear expression across tissues (UniProt Q12857; HPA tissue IHC). The separate subcellular dataset places NFIA mainly in the nucleoplasm and also lists cytosol, so record cytoplasmic staining separately rather than folding it into a nuclear score (HPA subcellular). Examine nuclei at high magnification alongside a hematoxylin counterstain, using the same visual threshold across matched sections (standard IHC practice). If staining appears exclusively cytoplasmic, check the no primary control and repeat the selected pH 8.0 retrieval before assigning it to NFIA (datasheet M03531; standard IHC practice).
Could NFIA isoforms or epitope accessibility explain inconsistent nuclear staining?
NFIA has 4 listed isoforms and multiple modified residues, including phosphoserines at positions 258 and 300 (UniProt Q12857). The supplied evidence does not locate the M03531 epitope or establish which isoforms it detects, so an isoform specific explanation for unequal staining remains unverified (record; datasheet M03531). Compare nuclear staining across sections processed with the same retrieval and detection settings before attributing differences to epitope availability (standard IHC practice). If isoform discrimination matters, obtain epitope mapping or an independently validated isoform specific reagent and document its validation separately (standard IHC practice).
How can IF help assess an unexpected NFIA IHC pattern?
In a complementary IF experiment, examine NFIA relative to a nuclear counterstain and a marker for the expected cell type; glial cells show high tissue staining in caudate and cerebral cortex (HPA tissue IHC). Choose a fluorophore channel after checking the specimen’s autofluorescence, and include single stain and no primary controls when assessing overlap (standard IF practice). NFIA lacks a transmembrane segment and is nuclear, so permeabilisation should allow antibody access to the nuclear compartment (UniProt Q12857; standard IF practice). Interpret IF overlap separately from chromogenic IHC intensity: the selected paraffin section image establishes an IHC staining condition, not an IF condition (datasheet M03531).
How do I separate NFIA signal from diffuse DAB background?
The selected paraffin section used 10% goat serum blocking, a biotinylated secondary antibody, and DAB detection (datasheet M03531). Compare a no primary section to identify signal from the detection system, and assess whether diffuse colour follows tissue edges or damaged areas (standard IHC practice). Include a peroxidase blocking step and check its effectiveness on control sections before interpreting brown deposits as antigen staining (standard chromogenic IHC practice). If background persists, optimise blocking and antibody concentration on adjacent sections while retaining EDTA retrieval at pH 8.0 as the reference condition (datasheet M03531; standard IHC practice). Count only well defined nuclear staining in intact cells (UniProt Q12857; standard IHC practice).
What is a defensible way to score NFIA staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, then count intact nuclei with signal above a threshold set using matched controls (UniProt Q12857; standard IHC practice). Report the percentage of positive nuclei and, if intensity is reproducible, an H-score calculated as the sum of percentage at each intensity multiplied by its 0–3 grade (standard IHC practice). For spatial comparisons, report positive nuclei per mm² of viable tissue and normalise counts to the total eligible nuclei or analysed area (standard IHC practice). Keep retrieval, detection, counterstain, imaging, and threshold settings consistent, and report cytoplasmic staining separately (datasheet M03531; HPA subcellular; standard IHC practice).
When should apparent NFIA positivity be treated as artefact?
A convincing result places signal in intact nuclei, consistent with NFIA’s nuclear annotation and the nuclear tissue IHC profile (UniProt Q12857; HPA tissue IHC). Check cell identity against morphology: high staining is reported in glial cells of caudate and cerebral cortex, but that distribution alone cannot identify every stained cell (HPA tissue IHC; standard IHC practice). Treat edge accentuation, necrotic deposits, and staining repeated in a no primary control as reasons to investigate artefact or endogenous enzyme activity (standard IHC practice). Recheck questionable areas on a serial section with the same pH 8.0 retrieval and score only reproducible nuclear signal in viable cells (datasheet M03531; standard IHC practice).
Boster reagents
Best NFIA / Nuclear factor 1 A-type IHC Antibodies
Validated anti-NFIA antibodies cover human intestinal cancer and tonsil paraffin sections (M03531 IHC captions), mouse and rat brain paraffin sections (PB10080 IHC captions), and A431 cell IF/ICC (M03531 IF caption).
Real IHC data
Anti-NFIA Antibody ® (monoclonal, 16H11)
Cat # M03531
Real IHC data
Anti-NFIA Antibody ®
Cat # PB10080
M03531 will render with human intestinal cancer paraffin-section IHC; its additional captions show human tonsil IHC and A431 cell IF/ICC (M03531 image captions). PB10080 will render with mouse brain paraffin-section IHC; an additional caption shows rat brain IHC (PB10080 IHC captions).
Which to pick: For human tissue IHC, choose monoclonal M03531, documented on paraffin sections at 1 μg/ml (M03531 catalog and IHC captions); for mouse or rat tissue IHC, choose rabbit PB10080, documented on paraffin sections at 2 μg/ml (PB10080 catalog and IHC captions). For IF/ICC, choose M03531, which lists IF and ICC and has an A431 cell IF image at 2 μg/ml (M03531 catalog and IF caption). PB10080 lists human, mouse and rat reactivity, though its IHC images show mouse and rat only (PB10080 catalog and IHC captions); fixative is unreported for both antibodies’ paraffin-section examples (M03531 and PB10080 IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q12857 (NFIA_HUMAN, Nuclear factor 1 A-type).
- Human Protein Atlas. NFIA tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. NFIA subcellular location (ICC-IF): Mainly localized to the nucleoplasm..
- Human Protein Atlas. NFIA antibody validation summary (2 antibodies).
- Molecular regulation of the developing commissural plate. The Journal of comparative neurology 2010 — PMC2910370.
- Identification and validation of NFIA as a novel prognostic marker in renal cell carcinoma. The journal of pathology. Clinical research 2023 — PMC10240150.
- Multiple intrinsic factors act in concert with Lhx2 to direct retinal gliogenesis. Scientific reports 2016 — PMC5015061.
- NFIA haploinsufficiency is associated with a CNS malformation syndrome and urinary tract defects. PLoS genetics 2007 — PMC1877820.
- PubMed PMID:10718198 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16710414 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NFIA (Nuclear factor 1 A-type). Expected localisation Nuclear staining across tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M03531). Validated IHC antibody: M03531 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adipose tissue (Adipocytes); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human intestinal cancer tissue; fixative not specified (datasheet M03531); 2. Fixation: Image fixative and duration unreported (datasheet M03531); 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 M03531); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M03531); 8. Primary antibody: Mouse monoclonal (clone 16H11) anti-NFIA, 0.5-1μg/ml (datasheet M03531); 9. Primary incubation: Overnight at 4 °C (datasheet M03531). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: Four isoforms; epitope effects are unknown (UniProt); Regulation: No specific expression regulator annotated (UniProt). Evidence: UniProt Q12857, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm., Datasheet M03531, PMC10240150. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NFIA/immunohistochemistry-ihc-guide.