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NFYB / Nuclear transcription factor Y subunit beta · IHC design guide
Design Immunohistochemistry for NFYB
Plan NFYB staining in paraffin sections using the observed ubiquitous nuclear tissue pattern (HPA tissue IHC). This guide covers fixation, staining controls and interpretation for chromogenic IHC.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NFYB 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 in tissue (HPA tissue IHC) | |
| Staining pattern | Ubiquitous nuclear staining across cell types (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A06714-2) | |
| 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 conditions consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining has medium concordance with RNA data (HPA tissue IHC) | |
| Regulation | Expression regulators not annotated (UniProt) | |
| Isoform / epitope | No annotated isoforms; one 1–207 chain (UniProt) |
Section 1
Recommended NFYB IHC & IF Protocols
The catalog antibody protocol uses Tris-EDTA retrieval (datasheet: pH 9.0). The published NFYB IHC protocols below provide colorectal cancer and Chinese alligator tissue examples (PMC12632091; PMC13274144).
| Sample | Paraffin-embedded human spleen tissue; fixative not specified (datasheet A06714-2) |
| Fixation | Image fixative and duration unreported (datasheet A06714-2); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Tris-EDTA pH 9.0 (datasheet A06714-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-NFYB, 1:100-1:300 (datasheet A06714-2) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | NFYB-positive staining in adipocytes of adipose tissue (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated Tris-EDTA retrieval at pH 9.0 for the catalog antibody (datasheet: A06714-2); optimize against the published citrate or microwave methods if needed (PMC12632091; PMC13274144).
Section 2
What Is the Expected NFYB Staining Pattern?
NFYB should appear predominantly in nuclei, consistent with its role as a nuclear transcription factor (UniProt P25208: nucleus). HPA describes ubiquitous nuclear expression and reports high staining in several cell populations, including adipocytes, glandular cells, hematopoietic cells and respiratory epithelial cells (HPA tissue IHC: High). The tissue profile is rated Supported, with medium consistency between antibody staining and RNA expression (HPA tissue IHC: Supported). NFYB has no transmembrane segment (UniProt P25208 topology).
| Clear nuclear staining in adipocytes, glandular cells, hematopoietic cells or respiratory epithelium. | This fits the reported broad nuclear pattern; these populations have High staining in the supplied tissue examples (HPA tissue IHC: High). Compare nuclei with neighboring cells on the same section rather than requiring identical intensity throughout the tissue. |
| Predominantly cytoplasmic, membranous or extracellular color, with little nuclear signal. | Treat this as a discordant compartment pattern and investigate artefact or nonspecific staining (UniProt P25208: nucleus; HPA tissue IHC: ubiquitous nuclear expression). NFYB has no annotated transmembrane segment or signal peptide to support a membrane or secreted pattern (UniProt P25208 topology and processing). |
| A cell population stains while the expected nuclear pattern is absent from nearby cells. | Check for cross-reactivity or endogenous detection activity before assigning the color to NFYB (HPA tissue IHC: ubiquitous nuclear expression; general IHC practice). Broad expression means an unlisted cell type alone is not evidence of a false positive; compartment and control results matter. |
| Weak, diffuse color covers nuclei and surrounding tissue without clear cell boundaries. | Background can obscure the nuclear result; assess it against a control omitting the primary antibody and review blocking and detection conditions (general IHC practice). A diffuse field cannot be scored as convincing NFYB localization solely because HPA reports broad expression (HPA tissue IHC: ubiquitous nuclear expression). |
| No nuclear signal in a supplied High-staining tissue example. | First check assay performance using the documented cell population, section quality, primary antibody and detection controls (HPA tissue IHC: High; general IHC practice). A failed positive control makes a negative study sample uninterpretable; the HPA profile is Supported, with medium staining–RNA consistency (HPA tissue IHC: Supported). |
💡Expected NFYB appearanceCall positive when distinct nuclear staining is visible in cells such as the listed High-staining adipocytes, glandular cells or hematopoietic cells (HPA tissue IHC: High; UniProt P25208: nucleus). Predominantly cytoplasmic or diffuse chromogen without convincing nuclei is a suspect positive pattern (HPA tissue IHC: ubiquitous nuclear expression).
| Tissue and cell context (HPA tissue IHC) | HPA reports ubiquitous nuclear expression and low tissue RNA specificity; its eight supplied High examples span adipocytes, glandular cells, hematopoietic cells, respiratory epithelium and neural cell populations (HPA tissue IHC: High; low tissue specificity). No supplied tissue is classified negative. |
| Evidence strength (HPA tissue IHC) | The tissue profile is Supported, with medium consistency between staining and RNA data (HPA tissue IHC: Supported). Use it as an expected pattern, and interpret an individual section alongside its controls. |
| Antibody validation (HPA antibodies) | HPA065646 and CAB004211 are each marked Supported for IHC and ICC (HPA antibodies: Supported). These entries support their reported applications but do not supply a dilution or an Enhanced IHC designation. |
| Protein organization (UniProt P25208) | NFYB is a nuclear NF-Y component that associates with NF-YC and NF-YA (UniProt P25208: subunit; nucleus). No transmembrane segment, signal peptide, propeptide or alternative isoform is annotated in this record (UniProt P25208: topology, processing, isoforms). |
| IF/ICC—where should fluorescence appear? | The expected location is nucleoplasm, reported as supported in HPA ICC-IF images (HPA subcellular: nucleoplasm, supported). This localization answers the IF/ICC pattern question; the primary application here is chromogenic IHC in paraffin sections. |
| Situation | Likely cause | Next action |
|---|---|---|
| The known-positive cell population has no nuclear stain. | The IHC run may have failed; loss of signal alone cannot establish absent NFYB where HPA reports High staining (HPA tissue IHC: High). | Check the documented positive tissue and assay controls, then review primary antibody use, retrieval and detection as general IHC workflow variables (general IHC practice). No NFYB-specific retrieval or fixation effect is established by these payloads. |
| Color is mainly cytoplasmic or outlines membranes. | The compartment conflicts with the reported nuclear localization (UniProt P25208: nucleus; HPA subcellular: nucleoplasm). | Review morphology and counterstain, compare a control omitting primary antibody, and repeat the assay if the mismatch persists (general IHC practice). Score convincing nuclei separately from off-target color. |
| Unexpected cells show isolated chromogen. | Cross-reactivity or endogenous detection activity is possible, although broad NFYB expression prevents labeling every unlisted cell type abnormal (HPA tissue IHC: ubiquitous nuclear expression; general IHC practice). | Confirm the stain is nuclear; inspect a control omitting primary antibody and apply the detection system's appropriate endogenous-activity control (general IHC practice). Compare with the supplied HPA cell examples. |
| Diffuse background makes nuclei hard to distinguish. | Background from blocking, reagent concentration or detection can reduce contrast in chromogenic IHC (general IHC practice). | Inspect a control omitting primary antibody, review blocking and antibody dilution, and compare nuclear detail with the counterstain (general IHC practice). No target-specific dilution is supplied. |
| Staining differs across fields or specimens. | The HPA tissue profile is Supported with medium staining–RNA consistency, and its supplied cell populations are reported as High rather than uniformly quantified (HPA tissue IHC: Supported; High). | Score nuclear localization and intensity by cell population; compare like regions and include the same positive control across runs (general IHC practice). Avoid inferring a negative tissue category from these data. |
| IF/ICC shows fluorescence outside the nucleus. | That distribution conflicts with the supported nucleoplasmic localization (HPA subcellular: nucleoplasm, supported). | Check the nuclear counterstain and appropriate fluorescence controls before assigning the signal to NFYB (general IF practice). Use the separate IF/ICC guide for assay setup. |
Experiment evidence
IHC & IF images showing NFYB staining
A06714-2 · IHCImmunohistochemical analysis of paraffin-embedded human spleen. 1, Tris-EDTA, pH9.0 was used for antigen retrieval. 2 Antibody was diluted a
A06714-3 · IHCImmunohistochemistry (IHC) analyzes of NF-YB (Q30) pAb in paraffin-embedded human breast carcinoma tissue at 1:100.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000120837-NFYB/tissue/cerebral+cortex#imid_1401428
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000120837-NFYB/tissue/colon#imid_1401354
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000120837-NFYB/tissue/liver#imid_1401347
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000120837-NFYB/tissue/kidney#imid_1401356
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB004211; green = NFYB, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB004211; green = NFYB, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NFYB IHC & IF
🧪Run bone marrow first: hematopoietic cells should show nuclear NFYB staining (HPA: High in hematopoietic cells; UniProt P25208: nucleus). HPA detects NFYB in all 45 scored tissues, so there is no HPA-supported negative tissue; use no-primary and isotype controls, and treat any cells with counterstained nuclei but no nuclear chromogen as observations rather than validated internal negatives (HPA: no negative tissue rows; standard IHC practice).
Positive control tissue: Adipose tissue (Adipocytes, HPA High)
Negative control tissue: None in HPA: NFYB 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 NFYB in A-431, U-251MG, U2OS, HAP1, SiHa, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only section, an isotype-matched control of the primary antibody’s host species and clonality, and, where available, an NFYB knockout specimen as a biological negative (standard IHC practice). Quench endogenous peroxidase in bone marrow, especially for chromogenic detection of hematopoietic cells (standard IHC practice; HPA: bone marrow hematopoietic cells).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the fixative is unreported in the selected A06714-2 paraffin-section caption (caption: fixative not stated). That caption uses Tris-EDTA at pH 9.0 for retrieval but does not establish that retrieval is required; the supplied evidence does not show that frozen sections or IF are easier (caption: retrieval condition; HPA: ICC-IF nucleoplasmic localisation). Endogenous peroxidase in bone marrow myeloid cells can produce misleading chromogenic signal unless quenched (standard IHC practice).
HPA tissue IHC evidence for NFYB
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 |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Adipocytes | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: NFYB is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced NFYB IHC Tips
Use nuclear staining as the primary readout for NFYB, and interpret tissue patterns alongside the stated antibody and reference evidence.
What retrieval should I try first for weak NFYB staining in paraffin sections?
Use heat-mediated Tris-EDTA at pH 9.0 first for paraffin-section NFYB IHC (datasheet A06714-2). The selected human spleen image used this retrieval, followed by catalog antibody at 1:200 overnight at 4°C (caption A06714-2). If nuclear staining remains weak, vary heating and cooling within your laboratory's validated retrieval workflow before considering another buffer or pH (general IHC practice). Compare each condition on adjacent sections with the same detection and counterstain, judging improvement by nuclear signal and preservation of tissue structure; the caption does not report the fixative (caption A06714-2; general IHC practice).
How should I troubleshoot fixation-related loss of NFYB staining?
Target-specific sensitivity of NFYB to fixation is unknown from the supplied evidence; the selected paraffin-section caption does not state a fixative (caption A06714-2). Record fixative, fixation duration, tissue thickness, and processing history for each specimen, since these variables can alter antigen accessibility in routine IHC (general IHC practice). Compare matched sections processed under documented conditions while keeping Tris-EDTA retrieval at pH 9.0 and the catalog antibody at 1:200 constant initially (datasheet A06714-2; general IHC practice). Judge changes against nuclear staining and tissue morphology rather than assigning any particular fixation effect to NFYB without direct evidence (UniProt P25208 subcellular location; general IHC practice).
What staining compartment should I expect, and how do I assess cytoplasmic signal?
Score NFYB primarily in nuclei: UniProt lists the nucleus, and the subcellular reference supports nucleoplasmic localisation (UniProt P25208 subcellular location; HPA subcellular: nucleoplasm supported). The tissue reference describes ubiquitous nuclear expression, so assess the proportion and intensity of stained nuclei within each defined cell population (HPA tissue IHC: ubiquitous nuclear expression). If cytoplasmic colour dominates, inspect an antibody omission control, counterstain quality, and whether pigment or precipitate extends beyond cell boundaries (general IHC practice). Reassess retrieval, antibody concentration, and detection background before treating cytoplasmic staining as a new NFYB distribution (datasheet A06714-2; general IHC practice).
Could an isoform or modified epitope explain inconsistent nuclear staining?
The supplied UniProt record lists 0 isoforms, a single chain spanning residues 1–207, and no annotated glycosylation sites or modified residues (UniProt P25208 processing, isoforms and annotations). Those annotations do not establish the catalog antibody's epitope or prove that processing cannot affect binding (UniProt P25208 annotations; general IHC practice). When results vary, check the antibody's stated immunogen or epitope information if available, then compare matched sections under the documented Tris-EDTA pH 9.0 retrieval (datasheet A06714-2; general IHC practice). Keep section handling and detection constant while assessing whether nuclear signal changes with retrieval or antibody concentration (general IHC practice).
How can I use IF to check the cell identity and location of an IHC signal?
On the separate IF/ICC workflow, combine NFYB with a validated adipocyte marker when examining adipose tissue, where the tissue reference reports high adipocyte staining (HPA tissue IHC: high in adipocytes; general IF practice). Choose spectrally separated fluorophores and consider a far-red channel if tissue autofluorescence interferes with shorter wavelengths (general IF practice). NFYB is nuclear and has no transmembrane segment, so permeabilisation should allow antibody access to the nuclear epitope; optimise it for the IF specimen and antibody (UniProt P25208 subcellular location and topology; general IF practice). Include single-channel and antibody omission controls before comparing IF localisation with the chromogenic nuclear readout (general IF practice).
How do I reduce diffuse colour or false nuclear staining?
First inspect an antibody omission control and tissue morphology to distinguish detection background from specific nuclear staining (general IHC practice; UniProt P25208 subcellular location). In chromogenic peroxidase IHC, use an appropriate endogenous peroxidase block and assess whether DAB colour appears in the control; these are general workflow checks, not NFYB-specific validation (general IHC practice). If background rises with signal, adjust blocking, washes, or primary antibody concentration around the caption's 1:200 starting point while keeping retrieval at pH 9.0 (caption A06714-2; datasheet A06714-2; general IHC practice). Review section edges and folds separately because concentrated chromogen there can mislead nuclear scoring (general IHC practice).
How should I quantify NFYB across tissue samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue area before scoring, then report the percentage of NFYB-positive nuclei and an intensity-based H-score when staining quality supports graded intensity (general IHC practice; UniProt P25208 subcellular location). Normalise positive-cell counts to all evaluable nuclei in that population, or report nuclear-positive density per mm² of evaluable tissue when cell density is the comparison of interest (general IHC practice). Apply one threshold, counterstain, and imaging procedure across specimens, and exclude folds, necrosis, and poorly preserved regions consistently (general IHC practice). Report background controls and the scoring rule alongside results because the tissue reference assigns staining only supported reliability (HPA tissue IHC: Supported; general IHC practice).
Which findings support a true NFYB-positive result rather than artefact?
A plausible result is nuclear staining in intact cells, consistent with the reported nuclear and supported nucleoplasmic localisation and ubiquitous nuclear tissue pattern (UniProt P25208 subcellular location; HPA subcellular: nucleoplasm supported; HPA tissue IHC: ubiquitous nuclear expression). Check that staining follows nuclei rather than section edges, folds, necrotic debris, or extracellular deposits, which can produce misleading chromogenic patterns (general IHC practice). Use an antibody omission control to identify detection or endogenous enzyme colour before assigning cells as positive (general IHC practice). Interpret intensity cautiously across tissues because the HPA tissue assessment is Supported with medium agreement between antibody staining and RNA expression (HPA tissue IHC: reliability description).
Boster reagents
Best NFYB / Nuclear transcription factor Y subunit beta IHC Antibodies
Both antibodies have human paraffin-section IHC images (catalog image captions) and list human, mouse, and rat reactivity (catalog reactivity); A06714-2 also lists IF (catalog applications).
Real IHC data
Anti-NF-YB Antibody
Cat # A06714-2
Real IHC data
Anti-NF-YB (Q30) Antibody
Cat # A06714-3
A06714-2 will render with human spleen paraffin-section IHC using Tris-EDTA retrieval at pH 9 and a 1:200 primary dilution (A06714-2 image caption). A06714-3 will render with human breast carcinoma paraffin-section IHC at 1:100 (A06714-3 image caption).
Which to pick: For tissue IHC, choose the spleen example with documented retrieval for A06714-2 or the breast carcinoma example for A06714-3 (catalog image captions). For IF/ICC planning, A06714-2 lists IF, while A06714-3 does not; neither has an IF image (catalog applications and image alts). Both are rabbit antibodies with listed human, mouse, and rat reactivity (catalog host and reactivity), but staining in each species needs validation; neither paraffin-section caption reports the fixative (catalog image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P25208 (NFYB_HUMAN, Nuclear transcription factor Y subunit beta).
- Human Protein Atlas. NFYB tissue IHC expression (reliability: Supported).
- Human Protein Atlas. NFYB subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. NFYB antibody validation summary (2 antibodies).
- Multi-omics analysis of the HMGB2(+) tumor epithelial cells in lactylation subgroups in colorectal cancer. Cell & bioscience 2025 — PMC12632091.
- Integrated Bioinformatics Analysis of Master Regulators in Anaplastic Thyroid Carcinoma. BioMed research international 2019 — PMC6512074.
- Dynamic changes in chromatin accessibility reveal the role of NF-Y targeting AURKB in mediating cell cycle during asynchronous oogenesis in the Chinese Alligator (Alligator sinensis). Frontiers in zoology 2026 — PMC13274144.
- PubMed PMID:1577736 — UniProt-cited evidence.
- PubMed PMID:1549471 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NFYB (Nuclear transcription factor Y subunit beta). Expected localisation Nuclear staining in tissue (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A06714-2). Validated IHC antibody: A06714-2 at 1:100-1:300 (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 workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human spleen tissue; fixative not specified (datasheet A06714-2); 2. Fixation: Image fixative and duration unreported (datasheet A06714-2); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: Tris-EDTA pH 9.0 (datasheet A06714-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-NFYB, 1:100-1:300 (datasheet A06714-2); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining has medium concordance with RNA data (HPA tissue IHC); Isoform / epitope: No annotated isoforms; one 1–207 chain (UniProt); Regulation: Expression regulators not annotated (UniProt). Evidence: UniProt P25208, HPA tissue IHC · Supported, HPA ICC-IF · nucleoplasm., Datasheet A06714-2, PMC12632091, PMC13274144. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NFYB/immunohistochemistry-ihc-guide.