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NFIB / Nuclear factor 1 B-type · IHC design guide
Design Immunohistochemistry for NFIB
Plan NFIB paraffin-section IHC around the general nuclear tissue pattern (HPA tissue IHC). This guide covers fixation, tissue controls and nuclear scoring, with epitope coverage to consider across six isoforms (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NFIB 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 | General nuclear staining (HPA tissue IHC) | |
| Staining pattern | Nuclear staining across multiple cell types (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A01537-1) | |
| Positive control | Heart muscle+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 expression regulator annotated (UniProt) | |
| Isoform / epitope | Six isoforms; check epitope coverage (UniProt) |
Section 1
Recommended NFIB IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: A01537-1) is accompanied by published NFIB staining protocols for mouse embryonic brain and human skin specimens (PMC2802587; PMC7723069).
| Sample | Paraffin-embedded human mammary cancer tissue; fixative not specified (datasheet A01537-1) |
| Fixation | Image fixative and duration unreported (datasheet A01537-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet A01537-1) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A01537-1) |
| Primary antibody | Rabbit anti-NFIB, 1μg/ml (datasheet A01537-1) |
| Primary incubation | Overnight at 4 °C (datasheet A01537-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A01537-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | NFIB-positive staining in cardiomyocytes of heart muscle (HPA tissue IHC: High). HPA tissue profile: General nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated citrate retrieval at pH 6 (datasheet: A01537-1); both published paraffin-section protocols also used citrate at pH 6 (PMC2802587; PMC7723069).
Section 2
What Is the Expected NFIB Staining Pattern?
NFIB is a nuclear, DNA-binding transcription factor with no transmembrane segment (UniProt O00712). In paraffin-section IHC, expect staining in nuclei across several cell types, with high staining reported in selected epithelial, alveolar and muscle cells (HPA: general nuclear expression; high in listed cells). HPA rates the tissue-IHC profile Supported, with medium consistency between antibody staining and RNA expression (HPA: tissue IHC).
| Distinct nuclear staining in pancreatic exocrine glandular cells or skin basal-layer cells, with a readable counterstain. | This fits reported high staining in those cells and the expected nuclear location (HPA: high in both cell groups; UniProt O00712: nucleus). Judge the cells individually: staining elsewhere in the section does not substitute for a nuclear signal in the intended cells (general IHC practice). |
| Strong staining chiefly in cytoplasm, at membranes, or throughout extracellular material while nuclei remain pale. | That compartment does not match NFIB's annotated nuclear location or the general nuclear tissue-IHC profile (UniProt O00712: nucleus; HPA: general nuclear expression). Treat it as suspect and check detection background and morphology before assigning it to NFIB (general IHC practice). |
| Prominent staining in a cell population outside the HPA high-staining examples, with little nuclear staining in the expected cells. | Investigate antibody cross-reactivity or endogenous detection activity (general IHC practice). HPA reports low tissue specificity and gives selected high and low examples, so an unlisted cell type alone cannot establish a false positive (HPA: low tissue specificity; tissue IHC). |
| A diffuse chromogenic haze covers nuclei and surrounding tissue, obscuring cell boundaries. | The background prevents a reliable nuclear call (general IHC practice; UniProt O00712: nucleus). Review blocking, wash and detection controls, then reassess whether discrete nuclei remain stained above the surrounding tissue (general IHC practice). |
| No nuclear staining appears in a section containing an HPA high-staining cell population. | A failed stain or sample-specific issue is possible; absence in one run does not establish absent NFIB expression (general IHC practice). Confirm the expected cell population and assay controls before interpreting the section (HPA: high in listed cell groups). |
💡Expected NFIB appearanceA positive IHC result shows discrete nuclear staining, potentially strong in HPA high-staining cells such as pancreatic exocrine glandular cells; dominant cytoplasmic haze or nuclear signal in every cell regardless of morphology is suspect (UniProt O00712: nucleus; HPA: high in pancreatic exocrine glandular cells; general IHC practice).
| Compartment and topology (UniProt O00712: nucleus; no transmembrane segment). | Score nuclei in intact, identifiable cells (general IHC practice). Membrane staining has no support from the supplied NFIB topology, while the HPA tissue profile describes general nuclear expression (UniProt O00712: no transmembrane segment; HPA: tissue IHC). |
| Choice of positive reference tissue (HPA: high in listed cell groups). | HPA reports high staining in cardiomyocytes, lung alveolar type I cells, ovarian follicle cells, pancreatic exocrine cells, and several glandular or skin basal-layer cells (HPA: tissue IHC). Pick a listed population whose morphology can be identified on the section (general IHC practice). |
| Lower-staining reference populations (HPA: low in listed cell groups). | HPA lists cells in testicular seminiferous ducts, smooth muscle, splenic red pulp and bone-marrow hematopoietic cells as low, with no negative examples supplied (HPA: tissue IHC). A low category is a relative comparison, not a guaranteed blank control (HPA: tissue IHC). |
| Strength of the tissue evidence (HPA: Supported; medium RNA–staining consistency). | Use the observed IHC pattern as a guide, not an absolute rule for every specimen (HPA: tissue IHC). The listed antibody HPA003956 has Supported IHC validation; the supplied record does not assign it Enhanced IHC validation (HPA: antibody validation). |
| Isoforms and epitope uncertainty (UniProt O00712: six isoforms). | The record lists isoforms 1–6 but supplies no antibody epitope map or isoform-specific IHC result (UniProt O00712; HPA: supplied antibody record). Do not infer that one isoform, phosphorylation state or retrieval condition explains a differing stain from these data. |
| IF/ICC Q: Where should NFIB appear? (HPA: subcellular ICC-IF). | A: HPA supports nucleoplasm and nucleoli fibrillar-center localization in ICC-IF images from A-431, U-251MG and U2OS (HPA: subcellular ICC-IF). This provides a localization cross-check; the supplied ICC-IF record does not define a paraffin-section IF protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| No stain in pancreatic exocrine cells or another identified HPA high-staining population (HPA: tissue IHC). | The assay or sample may have failed; this result alone does not identify which step is responsible (general IHC practice). | Check section integrity, primary-antibody inclusion and detection controls; then review retrieval and antibody concentration using the actual IHC protocol (general IHC practice). The supplied sources specify no NFIB dilution or retrieval setting. |
| Cytoplasmic or membrane signal dominates while nuclei are weak (UniProt O00712: nucleus). | Background or off-target binding is possible, but appearance alone cannot identify the mechanism (general IHC practice). | Inspect a control lacking primary antibody, verify nuclear counterstain and cell boundaries, and assess whether nuclear signal persists independently of diffuse color (general IHC practice). |
| Many unrelated structures share a uniform brown deposit (general IHC practice). | Endogenous peroxidase activity or nonspecific detection can contribute to chromogenic background (general IHC practice). | Review the detection-system controls, endogenous-enzyme blocking where applicable, wash steps and reagent exposure; compare with a section lacking primary antibody (general IHC practice). |
| The slide is faint despite identifiable HPA high-staining cells (HPA: tissue IHC). | Low assay sensitivity is possible, though the supplied sources do not establish NFIB-specific fixation sensitivity (general IHC practice; HPA: tissue IHC scope). | Verify counterstain, detection controls and the actual catalog antibody's IHC-P instructions before changing retrieval or dilution (general IHC practice). Do not assign a target-specific fixation cause from this record. |
| A low-staining reference population shows visible nuclear color (HPA: low in listed cell groups). | HPA's low category does not mean absent protein; staining also depends on how the local run is read (HPA: tissue IHC; general IHC practice). | Compare nuclear intensity and cell identity with a high-staining reference in the same run; avoid treating the low population as a mandatory negative control (HPA: tissue IHC; general IHC practice). |
| An unexpected cell population stains strongly while the reference cells do not (HPA: high in listed cell groups). | Possible cross-reactivity or assay background merits review; HPA's low tissue specificity also limits conclusions from cell identity alone (general IHC practice; HPA: tissue IHC). | Check the no-primary control, compartment and morphology, then repeat with appropriate assay controls before reporting the new cell pattern as NFIB (general IHC practice; UniProt O00712: nucleus). |
Experiment evidence
IHC & IF images showing NFIB staining
A01537-1 · IHCIHC analysis of NFIB/NF1B2 using anti-NFIB/NF1B2 antibody (A01537-1). NFIB/NF1B2 was detected in paraffin-embedded section of human mammary
A01537-1 · IHCIHC analysis of NFIB/NF1B2 using anti-NFIB/NF1B2 antibody (A01537-1). NFIB/NF1B2 was detected in paraffin-embedded section of mouse lung tis
M01537-1 · IFIF analysis of NFIB/NF1B2 using anti-NFIB/NF1B2 antibody (M01537-1). NFIB/NF1B2 was detected in an immunocytochemical section of A431 cells.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000147862-NFIB/tissue/cerebral+cortex#imid_1268652
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000147862-NFIB/tissue/colon#imid_1268594
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000147862-NFIB/tissue/liver#imid_1268587
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000147862-NFIB/tissue/kidney#imid_1268588
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA003956; green = NFIB, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA003956; green = NFIB, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NFIB IHC & IF
🧪Run heart muscle first: cardiomyocyte nuclei should stain (HPA: High in cardiomyocytes; UniProt O00712: nucleus). HPA lists no negative tissue and detects NFIB in all 45 scored tissues, so no-primary and isotype controls carry the negative; other cells on the heart section can show the local background level but should not be assumed NFIB-negative (HPA: no negative tissue rows).
Positive control tissue: Heart muscle (Cardiomyocytes, HPA High)
Negative control tissue: None in HPA: NFIB 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 NFIB in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (supported), Nucleoli fibrillar center (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only section and a concentration-matched rabbit isotype control for the rabbit catalog antibody; a validated NFIB knockout specimen or immunizing-peptide block can provide a biological specificity control (caption: rabbit anti-NFIB; standard IHC practice). Because the caption uses biotin-based detection with DAB, block endogenous peroxidase and check endogenous biotin background in heart tissue (caption: biotinylated secondary, SABC and DAB; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A01537-1 paraffin-section caption does not state its fixative (A01537-1 caption). The caption reports heat retrieval in citrate buffer at pH 6 for 20 minutes, but does not establish whether retrieval is required; frozen sections or IF cannot be judged easier from the supplied evidence (A01537-1 caption). For heart tissue, check biotin-based DAB background, especially when interpreting weak nuclear staining (caption: SABC and DAB; standard IHC practice).
HPA tissue IHC evidence for NFIB
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — 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 |
|---|---|---|---|---|
| Heart muscle | Cardiomyocytes | High | Protein (IHC) | HPA → |
| Lung | Alveolar cells type I | High | Protein (IHC) | HPA → |
| Ovary | Follicle cells | High | Protein (IHC) | HPA → |
| Pancreas | Exocrine glandular cells | High | Protein (IHC) | HPA → |
| Prostate | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: NFIB is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced NFIB IHC Tips
Troubleshoot NFIB staining in paraffin sections by checking nuclear localisation, processing conditions, cell identity, and matched controls before interpreting chromogenic signal.
What retrieval should I try first when NFIB nuclear staining is weak?
Use heat-mediated citrate buffer at pH 6 for antigen retrieval (datasheet A01537-1). The selected paraffin-section example heated sections for 20 minutes before staining with the catalog antibody (caption A01537-1). If nuclei remain weak, adjust heating or cooling time on matched sections while keeping tissue attachment and morphology intact; compare every condition against the same positive control. Score nuclear signal separately from diffuse cytoplasmic DAB, because NFIB is nuclear (UniProt O00712) and tissue IHC shows general nuclear expression (HPA: Supported). Record the retrieval condition alongside the antibody concentration so a stronger signal is not mistaken for a biological difference.
How should I troubleshoot weak NFIB staining when fixation details are uncertain?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative (caption A01537-1). Record the fixative, time to fixation, and fixation duration for each specimen, then compare sections processed together before changing antibody concentration. Uneven nuclear staining near section edges or damaged regions warrants inspection of morphology and processing records before assigning biological meaning. Keep retrieval at citrate pH 6 initially (datasheet A01537-1), and use a consistently stained control section to assess whether a processing change improves signal. NFIB is nuclear (UniProt O00712), but that localisation alone cannot establish its fixation tolerance.
Which staining compartment supports a positive NFIB call in chromogenic IHC?
Prioritise nuclear DAB signal within intact cells: NFIB is annotated in the nucleus (UniProt O00712), and tissue IHC reports general nuclear expression (HPA: Supported). The reported nucleoplasm and nucleolar fibrillar-centre locations come from subcellular IF data (HPA: subcellular), so routine chromogenic sections need not resolve those structures. Examine the hematoxylin counterstain to confirm that brown signal overlaps nuclei rather than adjacent cytoplasm or extracellular material. Compare expected cell populations on the same section; high staining is reported in lung alveolar type I cells and skin basal-layer cells (HPA: High). Diffuse cytoplasmic-only colour should prompt a specificity and detection-control review before scoring.
Could NFIB isoforms or epitope accessibility explain discordant nuclear staining?
NFIB has 6 annotated isoforms and multiple modified residues, including phosphoserine 264 and phosphothreonine 286 (UniProt O00712). Those annotations establish possible sources of molecular variation, but they do not identify the epitope recognised by A01537-1 or prove isoform-specific staining. Check the antibody's documented immunogen and epitope information before interpreting a negative section as absence of every NFIB isoform. Compare matched sections under the same citrate pH 6 retrieval condition (datasheet A01537-1), then test a retrieval adjustment if nuclear signal remains weak. Interpret any newly appearing cytoplasmic signal cautiously because NFIB's annotated location is nuclear (UniProt O00712).
How can IF help investigate an ambiguous NFIB IHC pattern?
On a matched section, multiplex NFIB with a validated marker for the cell population being examined and a nuclear counterstain; assess nuclear overlap within individual cells. NFIB is nuclear (UniProt O00712), while subcellular IF reports nucleoplasm and nucleolar fibrillar-centre staining (HPA: subcellular). Choose fluorophores after checking autofluorescence in the tissue and reserve a spectrally distinct channel for the weaker signal. Because NFIB has no annotated transmembrane segment (UniProt O00712), permeabilise sufficiently for antibody access to its intracellular nuclear epitope and compare permeabilised with minimally treated controls. Keep IF optimisation separate from the paraffin-section IHC conditions reported for A01537-1 (caption A01537-1).
What should I check when DAB obscures NFIB-positive nuclei?
The selected IHC example used a 10% goat-serum block, biotinylated secondary antibody, a streptavidin–biotin complex, and DAB (caption A01537-1). If staining is widespread, inspect no-primary and secondary-only controls, reduce excessive primary exposure, and verify that washing removes unbound reagent. Include a peroxidase block as a general chromogenic IHC step, and consider endogenous biotin when using a biotin-based detection system. Compare brown deposits with hematoxylin-defined nuclei, since NFIB is nuclear (UniProt O00712). If background persists, compare a biotin-free detection method on matched sections while retaining the citrate pH 6 retrieval condition (datasheet A01537-1).
How should I quantify NFIB across paraffin sections with variable cellularity? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and scoring region before reviewing intensity, then count only intact nuclei with credible NFIB signal (UniProt O00712: nucleus). Report the percentage of positive nuclei and, when intensity matters, an H-score calculated as the sum of each intensity category's percentage multiplied by its category value. Normalise counts to the number of eligible nuclei in each region; if reporting positive-cell density, divide by measured tissue area in mm². Apply identical retrieval, detection, and scoring thresholds across specimens, starting from citrate pH 6 retrieval (datasheet A01537-1). Document excluded folds, necrosis, and edges so processing artefacts do not shift the score.
How can I distinguish genuine NFIB staining from artefact in IHC?
A credible result shows signal in intact nuclei (UniProt O00712: nucleus) within the scored cell population and is reproducible across comparably processed sections. High staining has been reported in cardiomyocytes and pancreatic exocrine glandular cells, while low staining is reported in splenic red-pulp cells (HPA: tissue IHC); these patterns are context, not specimen-level proof. Review cytoplasmic-only deposits, section-edge accentuation, necrosis, and staining in unexpected cells against morphology and matched controls. A no-primary control helps identify detection-related colour, including endogenous peroxidase contribution to DAB signal. Treat discrepancies cautiously because the tissue IHC profile has Supported reliability with medium consistency between staining and RNA data (HPA: tissue IHC).
Boster reagents
Best NFIB / Nuclear factor 1 B-type IHC Antibodies
Anti-NFIB antibodies have IHC images from human mammary cancer, mouse lung and rat cardiac muscle paraffin sections, plus an IF image from A431 cells (catalog image captions).
Real IHC data
Anti-NFIB/NF1B2 Antibody ®
Cat # A01537-1
Real IF data
Anti-NFIB/NF1B2 Antibody ® (monoclonal, 4D6E4)
Cat # M01537-1
A01537-1 has IHC images from human mammary cancer, mouse lung and rat cardiac muscle paraffin sections (A01537-1 image captions). M01537-1 has an IF image from A431 cells and lists ICC and IF applications (M01537-1 image caption; catalog applications).
Which to pick: Choose rabbit polyclonal A01537-1 for tissue IHC; its images document citrate retrieval and chromogenic detection in paraffin sections across human, mouse and rat samples (A01537-1 image captions; catalog host). Choose mouse monoclonal M01537-1 for IF/ICC in A431 cells (M01537-1 image caption; catalog clone 4D6E4); for cross-species tissue IHC, A01537-1 has images for all three species (A01537-1 image captions). The A01537-1 captions identify paraffin sections but do not report the fixative (A01537-1 image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O00712 (NFIB_HUMAN, Nuclear factor 1 B-type).
- Human Protein Atlas. NFIB tissue IHC expression (reliability: Supported).
- Human Protein Atlas. NFIB subcellular location (ICC-IF): Localized to the nucleoplasm and nucleoli fibrillar center..
- Human Protein Atlas. NFIB antibody validation summary (1 antibodies).
- Nuclear factor IB is downregulated in vulvar squamous cell carcinoma (VSCC): Unravelling differentially expressed genes in VSCC through gene expression dataset analysis. Oncology letters 2021 — PMC8020388.
- Multiple non-cell-autonomous defects underlie neocortical callosal dysgenesis in Nfib-deficient mice. Neural development 2009 — PMC2802587.
- Nuclear factor I/B mediates epithelial-mesenchymal transition in human melanoma cells through ZEB1. Oncology letters 2021 — PMC7723069.
- Predictors of Outcome in Adenoid Cystic Carcinoma of Salivary Glands: A Clinicopathologic Study With Correlation Between MYB Fusion and Protein Expression. The American journal of surgical pathology 2017 — PMC5597477.
- PubMed PMID:9484777 — UniProt-cited evidence.
- PubMed PMID:9099724 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NFIB (Nuclear factor 1 B-type). Expected localisation General nuclear staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A01537-1). Validated IHC antibody: A01537-1 at 1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Heart muscle (Cardiomyocytes); 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 mammary cancer tissue; fixative not specified (datasheet A01537-1); 2. Fixation: Image fixative and duration unreported (datasheet A01537-1); 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: Citrate pH 6 , 20 min (datasheet A01537-1); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A01537-1); 8. Primary antibody: Rabbit anti-NFIB, 1μg/ml (datasheet A01537-1); 9. Primary incubation: Overnight at 4 °C (datasheet A01537-1). 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: Six isoforms ; check epitope coverage (UniProt); Regulation: No expression regulator annotated (UniProt). Evidence: UniProt O00712, HPA tissue IHC · Supported, HPA ICC-IF · nucleoplasm and nucleoli fibrillar center., Datasheet A01537-1, PMC2802587, PMC7723069. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NFIB/immunohistochemistry-ihc-guide.