NFKB2 / Nuclear factor NF-kappa-B p100 subunit · IHC design guide

Design Immunohistochemistry for NFKB2

Plan NFKB2 paraffin-section IHC around cytoplasmic staining and high expression in lymphoid tissues (HPA tissue IHC). Use the IHC-validated antibody at 0.5–1 μg/ml (datasheet PB9150), and consider p100-to-p52 processing when interpreting staining (UniProt).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for NFKB2 (IHC for NFKB2): expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC), antibody PB9150, validated IHC image, and IHC protocol steps — Printable **NFKB2** IHC protocol sheet — expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC), antibody PB9150, controls and protocol steps. Open the full NFKB2 IHC guide →

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NFKB2 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	Cytoplasmic staining in most tissues (HPA tissue IHC)
	Staining pattern	Cytoplasmic staining; highest in lymphoid tissues (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet PB9150)
	Positive control ⓘ	Appendix+4 more · see all
	Negative control ⓘ	Cerebellum+4 more · see all

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Keep fixation consistent across sections. (standard IHC practice; not target-specific)
	Caveat	A C-terminal epitope may miss processed p52 (UniProt)
	Regulation	Noncanonical signaling processes p100 (UniProt)
	Isoform / epitope	3 isoforms; p52 lacks the p100 C-terminus (UniProt)

Section 1

Recommended NFKB2 IHC & IF Protocols

The catalog antibody’s IHC-P protocol uses EDTA pH 8.0 heat retrieval (datasheet: PB9150). The published meningioma protocol below provides a citrate pH 6.0 alternative (PMC7445252).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded rat intestine tissue; fixative not specified (datasheet PB9150)
Fixation	Image fixative and duration unreported (datasheet PB9150); 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 PB9150); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet PB9150)
Primary antibody	Rabbit anti-NFKB2, 0.5-1μg/ml (datasheet PB9150)
Primary incubation	Overnight at 4 °C (datasheet PB9150)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9150)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	NFKB2-positive staining in lymphoid tissue of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues, highest expression in lymphoid tissues. No signal in the no-primary control.

💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet: PB9150); evaluate citrate pH 6.0 if adapting the published meningioma protocol (PMC7445252).

Section 2

What Is the Expected NFKB2 Staining Pattern?

NFKB2 is expected mainly in the cytoplasm across many tissues, with the strongest tissue profile in lymphoid tissue (HPA: Enhanced tissue IHC; cytoplasmic expression in most tissues). Nuclear staining can also fit its biology: inactive complexes can remain cytoplasmic, while active NF-κB complexes can enter nuclei (UniProt Q00653: subcellular location and function). NFKB2 has no transmembrane segment, so a crisp membrane rim is unexpected (UniProt Q00653: topology).

What am I looking at on my slide?
Cytoplasmic staining in lymphoid cells, including appendix lymphoid tissue and lymph node non-germinal center cells (HPA: High in both).	This fits the reported IHC profile; germinal center cells in tonsil are another high-staining reference (HPA: High in tonsil germinal center cells). Compare matched tissue compartments, since a whole-section score can hide differences between cell populations (standard IHC practice).
Nuclear staining accompanies cytoplasmic staining in some cells.	Nuclear localization is biologically plausible after pathway activation (UniProt Q00653: subcellular location and function). A single IHC image cannot establish that p100 was processed to p52 or that transcription is active (UniProt Q00653: p100/p52 processing; standard IHC interpretation).
A sharp membrane outline or extracellular deposit dominates the signal.	That compartment conflicts with the reported cytoplasmic and nuclear localization and the absence of a transmembrane segment (HPA: tissue IHC and subcellular profile; UniProt Q00653: topology). Treat it as suspect staining and check morphology and controls (standard IHC practice).
Strong signal appears in an unexpected cell population, such as cardiomyocytes or prostate glandular cells (HPA: Not detected in those cells).	The mismatch raises concern for antibody cross-reactivity or endogenous chromogen-generating activity, but a new biological context cannot be ruled out from this comparison alone (HPA: tissue IHC; standard IHC interpretation). Confirm cell identity and inspect detection controls (standard IHC practice).
The section shows diffuse haze, or a known high-staining compartment has no signal.	Diffuse haze limits compartment scoring and may reflect nonspecific staining or detection background (standard IHC practice). No signal in appendix lymphoid tissue or tonsil germinal center cells conflicts with their reported high staining; assess section quality and assay controls before calling the sample negative (HPA: High in both; standard IHC practice).

💡Expected NFKB2 appearanceCall a positive result when identifiable lymphoid cells show clear cytoplasmic staining, with possible nuclear signal, against a sufficiently clean background; appendix lymphoid tissue, lymph node non-germinal center cells and tonsil germinal center cells are reported High (HPA: tissue IHC; UniProt Q00653: subcellular location). A sharp membrane rim or staining confined to cells reported Not detected is a warning sign, not proof of a true positive (UniProt Q00653: topology; HPA: tissue IHC).

How each factor affects the staining
Cell population and tissue	HPA reports High staining in several lymphoid compartments, but also in placental decidual cells and testicular spermatogonia; its tissue profile has low RNA tissue specificity (HPA: tissue IHC and RNA specificity). Select and score the stated cells, not tissue names alone (standard IHC practice).
p100 processing and antibody epitope	NFKB2 p100 spans residues 1–900; p52 spans 1–454 (UniProt Q00653: chains). Whether an antibody detects both forms depends on its binding site, which is not supplied here; compartment staining alone cannot distinguish them (UniProt Q00653: processing; standard IHC interpretation).
Isoforms and validation	UniProt lists three isoforms, while HPA rates tissue IHC reliability Enhanced and lists two IHC Enhanced antibodies, HPA008422 and HPA023900 (UniProt Q00653: isoforms; HPA: antibodies and tissue IHC). Those ratings support the reported profile but do not establish which isoform a given antibody recognizes (HPA: validation; UniProt Q00653: isoforms).
Antigen retrieval	Retrieval conditions can affect staining in paraffin-section IHC (standard IHC practice). No NFKB2-specific retrieval requirement or fixation sensitivity is supplied by these records; evaluate conditions against positive tissue and background controls without assigning any change to NFKB2 epitope masking (standard IHC practice).
IF/ICC Q&A: Where should signal appear?	HPA reports enhanced nucleoplasm and cytosol localization in its ICC-IF images (HPA: subcellular profile). This supports interpreting those compartments in IF/ICC, but it does not supply an IF/ICC protocol option or convert an IF image into evidence of a particular IHC tissue pattern (HPA: subcellular and tissue profiles).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
No signal in appendix lymphoid tissue or tonsil germinal center cells (HPA: High in both).	The expected positive compartment may be absent from the section, or an IHC step may have failed; the image alone cannot distinguish these causes (HPA: tissue IHC; standard IHC practice).	Verify the relevant cells are present, then check the positive control, detection reagents and the antibody's validated IHC conditions (standard IHC practice).
Broad brown haze obscures cell boundaries.	Nonspecific antibody binding, excess detection signal or residual endogenous enzyme activity can raise chromogenic background (standard IHC practice).	Inspect the no-primary and detection controls; review blocking, washes, antibody concentration and endogenous enzyme blocking as applicable (standard IHC practice).
Most apparent signal forms crisp cell-membrane rims.	This distribution is discordant with NFKB2's reported cytoplasmic/nuclear location and lack of a transmembrane segment (HPA: localization; UniProt Q00653: topology).	Recheck cell borders against the counterstain and review negative controls; do not score the rim alone as NFKB2 positivity (standard IHC practice).
Strong staining occurs in cardiomyocytes or prostate glandular cells (HPA: Not detected in those cells).	Cross-reactivity or endogenous detection activity is possible; HPA's reference pattern does not identify the cause in this section (HPA: tissue IHC; standard IHC interpretation).	Confirm cell identity and compare no-primary and positive-tissue controls before reporting an unexpected positive population (standard IHC practice).
Nuclear signal is present, but cytoplasmic signal is weak.	Nuclear NFKB2 is plausible, yet compartment intensity alone cannot establish activation, processing or an assay fault (UniProt Q00653: subcellular location and processing).	Record nuclear and cytoplasmic staining separately; compare cell populations and controls before making a mechanistic claim (standard IHC practice).
Two lymphoid samples give different intensities despite comparable staining runs.	HPA reports High staining in specified cell populations, not a uniform intensity for every cell in each tissue (HPA: appendix, lymph node and tonsil IHC).	Score the named compartments separately and check that morphology, section quality and controls permit a fair comparison (standard IHC practice).

Source: UniProt Q00653 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing NFKB2 staining

IHC analysis of NFkB/NFKB2 p100/p52 using anti-NFkB/NFKB2 p100/p52 antibody (PB9150). NFkB/NFKB2 p100/p52 was detected in a paraffin-embedded section of rat intestine tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1 μg/ml rabbit anti-NFkB/NFKB2 p100/p52 Antibody (PB9150) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.

PB9150 · IHCIHC analysis of NFkB/NFKB2 p100/p52 using anti-NFkB/NFKB2 p100/p52 antibody (PB9150). NFkB/NFKB2 p100/p52 was detected in a paraffin-embedde

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000077150-NFKB2/tissue/cerebral+cortex#imid_2597096

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000077150-NFKB2/tissue/colon#imid_2597030

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000077150-NFKB2/tissue/liver#imid_6258823

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000077150-NFKB2/tissue/kidney#imid_2597016

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB022098; green = NFKB2, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for NFKB2 IHC & IF

🧪Run tonsil first; germinal center cells should stain (HPA: High in tonsil germinal center cells). Use heart muscle cardiomyocytes as the negative tissue (HPA: Not detected in cardiomyocytes); on the tonsil slide, assess morphologically identifiable unstained cells against background without assuming a particular cell type is negative.

Positive control tissue: Appendix (Lymphoid tissue, HPA High)

Negative control tissue: Cerebellum (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show NFKB2 in A-431, U-251MG, U2OS, hTERT-RPE1 (serum starved), with annotated localisation: Nucleoplasm (enhanced), Cytosol (enhanced) (HPA subcellular).

Technical controls: Include a no-primary (secondary-only) control, a concentration-matched rabbit IgG isotype control matched to the primary antibody’s clonality, and NFKB2 knockout material or validated immunizing-peptide competition as a biological specificity control (caption: rabbit primary; standard IHC practice). Quench endogenous peroxidase and check for endogenous biotin background when using the biotinylated secondary, SABC and DAB detection shown in the caption (caption: biotinylated secondary, SABC and DAB).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected PB9150 paraffin-section caption does not state the fixative (caption: fixative not stated). The caption uses heat retrieval in EDTA at pH 8.0, but does not establish whether retrieval is required under other conditions (caption: EDTA heat retrieval); comparative ease of frozen sections or IF is unreported. In densely cellular tonsil, overlapping cells can complicate nuclear-versus-cytoplasmic scoring (HPA: High in tonsil germinal center cells; UniProt Q00653: nucleus and cytoplasm).

HPA tissue IHC evidence for NFKB2

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data. Paired antibodies with high similarity supports the protein profile.). 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
Appendix	Lymphoid tissue	High	Protein (IHC)	HPA →
Lymph node	Non-germinal center cells	High	Protein (IHC)	HPA →
Placenta	Decidual cells	High	Protein (IHC)	HPA →
Testis	Spermatogonia cells	High	Protein (IHC)	HPA →
Tonsil	Germinal center cells	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Cerebellum	Cells in granular layer	Not detected	Protein (IHC)	HPA →
Epididymis	Glandular cells	Not detected	Protein (IHC)	HPA →
Heart muscle	Cardiomyocytes	Not detected	Protein (IHC)	HPA →
Liver	Cholangiocytes	Not detected	Protein (IHC)	HPA →
Ovary	Follicle cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced NFKB2 IHC Tips

Troubleshoot NFKB2 staining in paraffin sections by checking retrieval, compartment, cell identity and controls before interpreting signal intensity.

How should I retrieve NFKB2 in paraffin sections when staining is weak?

Start with heat-mediated retrieval in EDTA pH 8.0 for paraffin sections (datasheet PB9150). The demonstrated section used 1 μg/ml primary antibody overnight at 4°C, so check those conditions before changing retrieval (datasheet PB9150). If staining remains weak, compare a carefully controlled alternative retrieval buffer on adjacent sections, keeping detection and exposure conditions matched (standard IHC practice). Examine tissue morphology and background alongside signal, because excessive heating can damage sections and make staining harder to interpret (standard IHC practice). Include a lymphoid-rich positive control, since high staining is reported in appendix lymphoid tissue and tonsil germinal center cells (HPA: tissue IHC).

Could fixation explain weak or uneven NFKB2 staining?

NFKB2-specific sensitivity to fixation is unknown from the supplied evidence, and the paraffin-section caption does not report its fixative (datasheet PB9150). Record the actual fixative, fixation duration and section processing for each specimen before comparing staining intensity (standard IHC practice). If material is available, stain sections with different documented processing histories in the same run using the demonstrated EDTA pH 8.0 retrieval (datasheet PB9150; standard IHC practice). Compare nuclear detail, tissue integrity and nonspecific staining as well as NFKB2 signal (standard IHC practice). Do not treat a staining difference as a target-specific fixation effect without a controlled comparison (standard IHC practice).

Should NFKB2 staining be nuclear, cytoplasmic or both?

Evaluate nuclear and cytoplasmic staining separately: NFKB2 is reported in both compartments, with inactive complexes retained in the cytoplasm (UniProt Q00653: subcellular location and function). The tissue IHC profile describes predominantly cytoplasmic expression in most tissues, with the highest expression in lymphoid tissues (HPA: tissue IHC). Nuclear signal can be biologically plausible because processed p52 participates in transcriptional complexes, but location alone does not establish processing or activation (UniProt Q00653: processing and function). Use a counterstain to define nuclei and score only intact cells with interpretable boundaries (standard IHC practice). Compare compartment patterns against a matched positive control and negative detection control before assigning biological meaning (standard IHC practice).

Can this stain distinguish full-length p100 from processed p52?

Do not assign a chromogenic signal to p100 or p52 without knowing the antibody epitope (UniProt Q00653: processing; standard IHC interpretation). Full-length p100 spans residues 1–900, while processed p52 spans 1–454 (UniProt Q00653: chains). An epitope within the shared N-terminal region could detect both forms; an epitope confined to the C-terminal region would exclude p52 (UniProt Q00653: chains; epitope-dependent inference). The record also lists three isoforms, so establish which sequences the antibody recognizes before making isoform claims (UniProt Q00653: isoforms; standard assay validation). Score nuclear and cytoplasmic staining descriptively until epitope mapping or another form-specific assay supports attribution (standard IHC practice).

How can I extend the NFKB2 readout to multiplex IF?

Plan IF as a separate assay and verify its fixation and retrieval conditions independently of the paraffin-section IHC caption (datasheet PB9150; standard IF practice). Multiplex NFKB2 with a validated marker identifying the cell population of interest, such as a lymphoid cell marker when assessing lymphoid tissue (HPA: tissue IHC; standard IF practice). Choose spectrally separated fluorophores after checking tissue autofluorescence, and include single-stain and no-primary controls for bleed-through and background (standard IF practice). Because NFKB2 is found in nucleoplasm and cytosol and has no transmembrane segment, permeabilise sufficiently to access intracellular epitopes while preserving morphology (HPA: subcellular; UniProt Q00653: topology; standard IF practice).

What should I check when NFKB2 DAB staining is diffuse?

First compare the stained section with a no-primary control to separate antibody-associated signal from detection background (standard IHC practice). The demonstrated workflow used 10% goat serum blocking, a biotinylated secondary, streptavidin-biotin detection and DAB (datasheet PB9150). Check peroxidase blocking and assess endogenous biotin when using that detection scheme; these are general chromogenic IHC controls (standard IHC practice). Diffuse cytoplasmic staining is not automatically artefactual because the tissue profile reports cytoplasmic NFKB2 in most tissues (HPA: tissue IHC). Reject staining concentrated at section edges, folds or damaged areas when the same pattern appears in controls (standard IHC practice).

How should I score NFKB2 staining across specimens? ⚠ ANSWER MARKED FOR VERIFICATION

Define the tissue region and cell population before scoring, then keep segmentation and intensity thresholds consistent across specimens (standard IHC practice). Report an H-score from the percentage of cells in each intensity category, or report % positive cells with nuclear and cytoplasmic results separated (standard IHC practice). For spatial questions, count positive cells per mm² of evaluable tissue and report the corresponding total cell density (standard IHC practice). Normalise to the number of eligible cells or evaluable tissue area, and exclude necrosis, folds and section edges consistently (standard IHC practice). Document the positive threshold against controls because baseline cytoplasmic expression varies by tissue (HPA: tissue IHC; standard IHC practice).

How do I distinguish meaningful NFKB2 staining from artefact?

A plausible positive should occupy intact cells in a biologically credible compartment and persist away from section edges and damaged tissue (UniProt Q00653: subcellular location; standard IHC practice). High staining in appendix lymphoid tissue or tonsil germinal center cells offers a supported positive reference (HPA: tissue IHC). Review unexpected staining in cell populations reported as undetected, such as heart muscle cardiomyocytes, alongside morphology and controls (HPA: tissue IHC; standard IHC practice). Edge staining, necrotic regions or signal reproduced by a no-primary control warrants investigation of nonspecific detection or endogenous enzyme activity (standard IHC practice). Nuclear staining alone cannot prove p100 processing to p52 or pathway activation without additional evidence (UniProt Q00653: processing and function; standard IHC interpretation).

Boster reagents

Best NFKB2 / Nuclear factor NF-kappa-B p100 subunit IHC Antibodies

The catalog includes paraffin-section IHC images from rat and mouse intestine and human lung and mammary cancer samples (PB9150 image captions); IF and ICC appear in antibody application lists (catalog applications).

Real IHC data

Anti-NFkB/NFKB2 p100/p52 Antibody ®

Cat # PB9150

View product →

PB9150 will render with its rat intestine paraffin-section IHC figure (PB9150 figure caption). Its additional IHC captions document human lung cancer, human mammary cancer and mouse intestine paraffin sections; its catalog lists Human, Mouse and Rat reactivity (PB9150 image captions; catalog reactivity).

Which to pick: Choose PB9150 for tissue IHC: its own captions document paraffin-section staining, but do not report the fixative (PB9150 image captions). For IF/ICC, M01228-1 lists both applications, while M01228-2 lists IF only; neither has an IF image in the supplied catalog (catalog applications and image alts). All three SKUs list Human, Mouse and Rat reactivity; PB9150 has paraffin-section IHC images across those species, while M01228-1 and M01228-2 are rabbit monoclonals (catalog reactivity and clone fields; PB9150 image captions).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry Q00653 (NFKB2_HUMAN, Nuclear factor NF-kappa-B p100 subunit).
Human Protein Atlas. NFKB2 tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. NFKB2 subcellular location (ICC-IF): Localized to the nucleoplasm and cytosol..
Human Protein Atlas. NFKB2 antibody validation summary (3 antibodies).
Identification of Prognostic Markers of Gynecologic Cancers Utilizing Patient-Derived Xenograft Mouse Models. Cancers 2022 — PMC8834149.
Developing and experimentally validating a glucocorticoid signaling-related gene signature to evaluate the prognosis and immunotherapeutic response in kidney renal clear cell carcinoma. PloS one 2025 — PMC12517536.
Programmed death ligand-1 (PD-L1) expression in meningioma; prognostic significance and its association with hypoxia and NFKB2 expression. Scientific reports 2020 — PMC7445252.
Ligand-based design and synthesis of N'-Benzylidene-3,4-dimethoxybenzohydrazide derivatives as potential antimicrobial agents; evaluation by in vitro, in vivo, and in silico approaches with SAR studies. Journal of enzyme inhibition and medicinal chemistry 2022 — PMC9037180.
PubMed PMID:1876189 — UniProt-cited evidence.
PubMed PMID:1531086 — UniProt-cited evidence.
PubMed PMID:8036016 — UniProt-cited evidence.