NFKBIA / NF-kappa-B inhibitor alpha · IHC design guide

Design Immunohistochemistry for NFKBIA

This guide covers paraffin-section chromogenic IHC for NFKBIA, with cytoplasmic staining expected in most tissues (HPA tissue IHC). It addresses stimulation-dependent degradation (UniProt) and the catalog antibody’s 1:50 IHC dilution (datasheet M01139).

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

Immunohistochemistry protocol sheet for NFKBIA (IHC for NFKBIA): expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC), antibody M01139, validated IHC image, and IHC protocol steps — Printable **NFKBIA** IHC protocol sheet — expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC), antibody M01139, controls and protocol steps. Open the full NFKBIA IHC guide →

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NFKBIA 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 signal in glandular and hematopoietic cells (HPA tissue IHC)
	Antigen retrieval	Citrate pH 6 HIER, heat-mediated (datasheet M01139)
	Positive control ⓘ	Adrenal gland+4 more · see all
	Negative control ⓘ	Cervix+4 more · see all

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M01139)
	Caveat	Immune activation may reduce signal through degradation (UniProt)
	Regulation	Inflammatory stimulation drives degradation (UniProt)
	Isoform / epitope	One 1–317 chain; no isoforms or processing annotated (UniProt)

Section 1

Recommended NFKBIA IHC & IF Protocols

The catalog antibody’s IHC-P protocol (datasheet M01139) is accompanied by published NFKBIA chromogenic IHC methods for glioma and mouse myocardial sections (PMC4199416; PMC7176878).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded mouse stomach tissue; fixative not specified (datasheet M01139)
Fixation	Image fixative and duration unreported (datasheet M01139); 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 M01139)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet M01139)
Primary antibody	Rabbit monoclonal (clone HO-14) anti-NFKBIA, 1:50 recommended; image 1ug/ml (datasheet M01139)
Primary incubation	Overnight at 4 °C (datasheet M01139)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet M01139)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	NFKBIA-positive staining in glandular cells of adrenal gland (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control.

💡Decision noteStart with heat-mediated citrate retrieval at pH 6 for the catalog antibody (datasheet M01139); the glioma protocol also used citrate at pH 6 (PMC4199416).

Section 2

What Is the Expected NFKBIA Staining Pattern?

NFKBIA should show predominantly cytoplasmic staining in many cell types, including glandular and hematopoietic cells that HPA rates Medium (HPA: cytoplasmic expression in most tissues; Medium in appendix glandular cells and bone marrow hematopoietic cells). Nuclear staining may occur because the protein shuttles between cytoplasm and nucleus (UniProt P25963: subcellular location). NFKBIA has no transmembrane segment, so a membrane rim is not expected (UniProt P25963 topology). HPA rates its tissue IHC evidence Approved, with medium consistency between staining and RNA data (HPA: tissue IHC reliability).

What am I looking at on my slide?
Cytoplasmic chromogen in the expected cells, with distinguishable unstained areas.	This fits the predominant tissue pattern (HPA: cytoplasmic expression in most tissues). Medium staining is reported in appendix glandular cells and bone marrow hematopoietic cells (HPA: tissue IHC). Judge each cell population separately; HPA levels describe observations, not an intensity threshold that every section must reproduce.
A sharp membrane rim, or strong nuclear-only staining across most cells.	A membrane rim conflicts with the lack of a transmembrane segment (UniProt P25963 topology). Widespread nuclear-only staining departs from HPA's mainly cytosolic IF pattern and cytoplasmic tissue profile (HPA: subcellular IF; tissue IHC). Review the staining pattern and controls before calling it NFKBIA; some nuclear localization remains biologically possible (UniProt P25963: nuclear–cytoplasmic shuttling).
Strong signal in a cell population HPA lists as Not detected.	For example, HPA reports kidney glomerular cells and liver cholangiocytes as Not detected (HPA: tissue IHC). Check cell identity and a negative control; antibody cross-reactivity or endogenous chromogenic activity can mimic target staining (general IHC practice). A negative listing for one cell population does not describe the entire organ (HPA: tissue IHC).
Diffuse color over stroma, lumina, and cells, without clear cellular borders.	This is difficult to interpret as the reported cytoplasmic pattern (HPA: tissue IHC). Background may reflect nonspecific binding, residual endogenous detection activity, or excessive development (general IHC practice). Compare a section processed without primary antibody and review the detection and development steps before scoring cells (general IHC practice).
No signal in a section containing expected positive cells.	First confirm that the section includes the relevant cells: HPA reports Medium staining in breast glandular cells and bone marrow hematopoietic cells (HPA: tissue IHC). Then check the catalog antibody's IHC-P instructions, detection reagents, and a positive control (general IHC practice). An absent signal alone cannot establish absence of NFKBIA.

💡Expected NFKBIA appearanceA positive IHC result is predominantly cytoplasmic staining in identifiable glandular or hematopoietic cells, with Medium staining reported for several such populations; a crisp membrane rim or indiscriminate background should prompt review (HPA: tissue IHC; UniProt P25963 topology).

How each factor affects the staining
Compartment and cell selection	HPA reports cytoplasmic expression in most tissues, but levels vary by named cell population (HPA: tissue IHC). Use appendix glandular cells or bone marrow hematopoietic cells as examples of reported Medium staining; kidney glomerular cells are listed as Not detected (HPA: tissue IHC).
Biological movement	NFKBIA shuttles between cytoplasm and nucleus, and stimulation promotes phosphorylation and degradation while allowing RELA nuclear entry (UniProt P25963: subcellular location and function). Interpret unusual nuclear staining with the experiment's stimulation context; staining alone cannot establish pathway activation.
Protein architecture	The recorded chain spans residues 1–317, with no signal peptide, propeptide, or transmembrane segment (UniProt P25963 topology and processing). No supplied evidence supports a shed or membrane-bound staining pattern. The supplied record has no mapped antibody epitope, so epitope-specific retrieval predictions are unavailable.
Evidence strength	HPA rates tissue IHC Approved and describes medium consistency with RNA data; the listed antibodies are IHC Approved, while their ICC ratings differ (HPA: tissue IHC reliability; HPA: antibodies). Treat the tissue profile as a guide to expected staining, not proof that every specimen or antibody will match it.
IF/ICC expectation?	Mainly cytosolic signal is the HPA IF expectation; A-431, U-251MG, U2OS, and HeLa BAC 6210 have ICC-IF images (HPA: subcellular IF). Nuclear localization remains possible through shuttling (UniProt P25963: subcellular location). IF/ICC optimization belongs in its separate guide; these observations do not define an IHC-P protocol.
Retrieval and detection	No target-specific fixation sensitivity or retrieval condition is supplied (HPA: tissue IHC; UniProt P25963). Select retrieval and chromogenic detection conditions from the IHC-validated antibody's instructions, then assess controls together with the specimen (general IHC practice).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Positive control stains, but the study section appears negative.	The sampled area may lack the cell population being assessed, or its staining may be below visual detection; HPA levels are cell-specific observations (HPA: tissue IHC).	Verify tissue and cell identity on the counterstain, then compare like cell populations and exposure to chromogen with the positive control (general IHC practice).
Expected positive tissue and study section are both blank.	A shared staining-step or detection failure is possible; HPA reports Medium staining in bone marrow hematopoietic cells and appendix glandular cells (HPA: tissue IHC).	Check the catalog antibody's IHC-P instructions, reagent activity, incubation steps, and chromogen development; rerun with an appropriate positive section (general IHC practice).
Color appears in both the stained section and the no-primary control.	The result may arise from endogenous detection activity or nonspecific detection reagents rather than primary-antibody binding (general IHC practice).	Review the relevant endogenous-activity block and detection controls before changing the primary-antibody conditions (general IHC practice).
Nuclear staining dominates and cytoplasm is nearly blank.	This differs from HPA's predominant cytoplasmic tissue pattern, although NFKBIA can shuttle into nuclei (HPA: tissue IHC; UniProt P25963: subcellular location).	Review stimulation context, cell identity, and controls; avoid assigning pathway activation solely from the IHC compartment pattern (UniProt P25963: function; general IHC practice).
A continuous membrane rim or staining of an unexpected cell population dominates.	A membrane rim conflicts with NFKBIA topology; unexpected cells may reflect cross-reactivity or chromogenic background (UniProt P25963 topology; general IHC practice).	Compare cellular morphology and no-primary controls, and assess the pattern against HPA's cell-specific observations before scoring it as positive (HPA: tissue IHC; general IHC practice).
The section has widespread haze that obscures cell boundaries.	Nonspecific binding or excess chromogen development can obscure a cytoplasmic pattern (general IHC practice; HPA: tissue IHC profile).	Review blocking, washing, antibody concentration, and development time using the catalog antibody's IHC-P instructions; retain a matched control for comparison (general IHC practice).

Source: UniProt P25963 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing NFKBIA staining

Immunohistochemical analysis of paraffin-embedded mouse stomach, using IKB alpha Antibody(M01139) NFKBIA was detected in paraffin-embedded tissue section. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1ug/ml rabbit anti-NFKBIA Antibody (M01139)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.

M01139 · IHCImmunohistochemical analysis of paraffin-embedded mouse stomach, using IKB alpha Antibody(M01139) NFKBIA was detected in paraffin-embedded t

M01139 · IFImmunofluorescent analysis of Hela cells, using IKB alpha Antibody .

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000100906-NFKBIA/tissue/cerebral+cortex#imid_1223192

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000100906-NFKBIA/tissue/colon#imid_1223118

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000100906-NFKBIA/tissue/liver#imid_1223119

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000100906-NFKBIA/tissue/kidney#imid_1223128

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA029207; green = NFKBIA, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for NFKBIA IHC & IF

🧪Run adrenal gland first and assess staining in its glandular cells (Medium; HPA: adrenal gland, glandular cells). Use cervix glandular cells as the negative tissue (HPA: Not detected); on the positive slide, treat cells without detectable signal as internal background only if they are observed, without assuming a particular cell type is negative.

Positive control tissue: Adrenal gland (Glandular cells, HPA Medium)

Negative control tissue: Cervix (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show NFKBIA in A-431, U-251MG, U2OS, HeLa BAC 6210, with annotated localisation: Cytosol (enhanced) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control, a host- and clonality-matched isotype control (rabbit host; selected-SKU caption), and a biological negative such as NFKBIA knockout tissue or a validated peptide-block control. Check endogenous peroxidase and biotin background in the adrenal section because the selected-SKU caption uses biotin-based detection with DAB (selected-SKU caption).

⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected-SKU paraffin-section caption does not state a fixative (selected-SKU caption). That caption uses heat retrieval in citrate buffer at pH 6 for 20 minutes, but does not establish that retrieval is required (selected-SKU caption). The supplied evidence does not establish whether frozen sections or IF are easier; for adrenal IHC, assess peroxidase and biotin background with the stated detection method (selected-SKU caption).

HPA tissue IHC evidence for NFKBIA

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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
Adrenal gland	Glandular cells	Medium	Protein (IHC)	HPA →
Appendix	Glandular cells	Medium	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	Medium	Protein (IHC)	HPA →
Breast	Glandular cells	Medium	Protein (IHC)	HPA →
Bronchus	Respiratory epithelial cells	Medium	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Cervix	Glandular cells	Not detected	Protein (IHC)	HPA →
Esophagus	Squamous epithelial cells	Not detected	Protein (IHC)	HPA →
Kidney	Cells in glomeruli	Not detected	Protein (IHC)	HPA →
Liver	Cholangiocytes	Not detected	Protein (IHC)	HPA →
Oral mucosa	Squamous epithelial cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced NFKBIA IHC Tips

Troubleshoot NFKBIA staining in paraffin sections by checking retrieval, cell compartment, controls and scoring against the documented IHC evidence.

How should I adjust retrieval when NFKBIA staining is weak or uneven?

Start with heat-mediated citrate retrieval at pH 6 for 20 minutes (datasheet M01139). The selected paraffin-section example then used 10% goat serum and 1 µg/mL primary antibody overnight at 4 °C, so check those steps before changing retrieval (datasheet M01139). If staining remains weak, compare a small retrieval time series while holding antibody concentration, section thickness and detection constant; examine tissue damage as well as signal (standard IHC practice). Treat a new buffer or pH as an optimization experiment and retain a consistently stained reference section in each run (standard IHC practice).

Could fixation explain faint or patchy NFKBIA staining?

Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative, and its retrieval result cannot establish one (datasheet M01139). Record the fixative, fixation duration and tissue-processing history for each section before comparing NFKBIA intensity across specimens (standard IHC practice). If staining varies, compare similarly processed sections using the documented citrate pH 6, 20-minute retrieval and 1 µg/mL primary incubation as a common starting point (datasheet M01139; standard IHC practice). Use morphology and a consistently processed reference section to judge whether a processing difference, rather than biological variation, tracks with the signal (standard IHC practice).

How should I troubleshoot unexpectedly nuclear NFKBIA staining?

NFKBIA is found in cytoplasm and nucleus and shuttles through nuclear import and CRM1-dependent export, so some nuclear staining is biologically possible (UniProt P25963 subcellular). Predominantly cytoplasmic tissue staining is the reported reference pattern, while enhanced cytosolic localization comes from cell imaging (HPA tissue IHC; HPA subcellular). Check whether nuclear signal lies inside intact counterstained nuclei and occurs reproducibly in identifiable cells, rather than at section edges or damaged areas (standard IHC practice). Score nuclear and cytoplasmic staining separately, using the same retrieval and detection settings across compared sections; location alone does not establish pathway activation (standard IHC practice; UniProt P25963 function).

Can epitope choice change how I interpret NFKBIA IHC?

The supplied record lists one NFKBIA chain, residues 1–317, and 0 annotated isoforms; it does not establish which epitope the catalog antibody recognizes (UniProt P25963 processing and isoforms; datasheet M01139). NFKBIA has no transmembrane segment, so membrane-restricted staining would need careful validation before assignment to this target (UniProt P25963 topology). Phosphorylation is annotated at residues 32 and 36, among other modifications, but total-antibody staining cannot identify a modification without epitope-specific evidence (UniProt P25963 modified residues; standard IHC practice). When comparing antibodies, document their stated epitopes and compare cell-level patterns under matched section processing and controls (standard IHC practice).

How can I use IF to investigate an ambiguous IHC pattern?

Use IF/ICC as a separate validation experiment and pair NFKBIA with a marker for the cell population under study; the supplied HPA cell images report mainly cytosolic NFKBIA (HPA subcellular; standard IF practice). Choose spectrally separated fluorophores after checking the tissue's autofluorescence, and include single-stain controls to assess bleed-through (standard IF practice). Because NFKBIA lacks a transmembrane segment and is reported in cytoplasm and nucleus, use fixation-compatible permeabilisation that permits access to intracellular epitopes (UniProt P25963 topology and subcellular; standard IF practice). Compare nuclear and cytoplasmic signals with a nuclear counterstain, while keeping IF findings distinct from the catalog antibody's paraffin-section evidence (standard IF practice; datasheet M01139).

What should I check when DAB staining obscures NFKBIA-positive cells?

The selected IHC example used a biotinylated secondary, streptavidin–biotin complex and DAB, so investigate background at each of those detection stages (datasheet M01139; standard IHC practice). Include a primary-omission control and assess endogenous peroxidase and endogenous biotin contributions before attributing diffuse brown signal to NFKBIA (standard IHC practice). Check whether the 10% goat-serum block, 1 µg/mL primary concentration and overnight incubation at 4 °C reproduce the documented starting conditions (datasheet M01139). Compare background in intact central tissue with section edges and damaged areas, and shorten chromogen development only after verifying that the positive reference remains detectable (standard IHC practice).

How should I quantify NFKBIA staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and compartment before scoring, because NFKBIA can occupy cytoplasm and nucleus while tissue IHC is reported mainly as cytoplasmic (UniProt P25963 subcellular; HPA tissue IHC). For each compartment, report percent positive cells and an intensity-weighted H-score from 0–300, or positive-cell density per mm² when cell counts are appropriate (standard IHC practice). Normalize counts to the number of eligible intact cells, or density to analyzed tissue area, and apply identical thresholds and exclusion rules across slides (standard IHC practice). Keep retrieval, DAB development and image capture consistent, and report the reference section used to monitor run-to-run variation (standard IHC practice).

Which findings support a true NFKBIA-positive IHC result?

A reproducible intracellular pattern is plausible for NFKBIA, which has no transmembrane segment and is reported in cytoplasm and nucleus (UniProt P25963 topology and subcellular). Compare candidate positive cells with the expected cytoplasmic tissue pattern and with cell populations that have documented HPA staining, such as bone-marrow hematopoietic cells; HPA rates its tissue IHC evidence Approved with medium RNA concordance (HPA tissue IHC). Question staining confined to membranes, section edges or necrotic areas, or reproduced in a primary-omission control, before scoring it as positive (UniProt P25963 topology; standard IHC practice). Because the documented workflow uses DAB, check endogenous enzyme background when diffuse brown signal lacks a clear cellular boundary (datasheet M01139; standard IHC practice).

Boster reagents

Best NFKBIA / NF-kappa-B inhibitor alpha IHC Antibodies

M01139 has IHC data from paraffin-embedded mouse stomach (IHC image caption) and IF data from HeLa cells (IF image caption); listed reactivity covers human, mouse and rat (catalog reactivity).

Real IHC data

Anti-IKB alpha NFKBIA Rabbit Monoclonal Antibody

Cat # M01139

View product →

M01139 is shown in IHC on paraffin-embedded mouse stomach (IHC image caption). M01139 is also shown in IF on HeLa cells (IF image caption) and is listed for IHC, IF and ICC (catalog applications).

Which to pick: For tissue IHC, choose M01139: its IHC image shows staining in a paraffin-embedded mouse stomach section (IHC image caption); the fixative is unreported (IHC image caption). For IF/ICC, M01139 is listed for both applications (catalog applications), with an IF image from HeLa cells (IF image caption). For human, mouse or rat samples, M01139 is the listed rabbit monoclonal option (catalog host and clone; catalog reactivity); the IHC image specifically documents mouse tissue (IHC image caption).

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

References

UniProt Consortium. UniProt entry P25963 (IKBA_HUMAN, NF-kappa-B inhibitor alpha).
Human Protein Atlas. NFKBIA tissue IHC expression (reliability: Approved).
Human Protein Atlas. NFKBIA subcellular location (ICC-IF): Mainly localized to the cytosol..
Human Protein Atlas. NFKBIA antibody validation summary (2 antibodies).
Identification of a NFKBIA polymorphism associated with lower NFKBIA protein levels and poor survival outcomes in patients with glioblastoma multiforme. International journal of molecular medicine 2014 — PMC4199416.
Silencing of ATP2B1-AS1 contributes to protection against myocardial infarction in mouse via blocking NFKBIA-mediated NF-κB signalling pathway. Journal of cellular and molecular medicine 2020 — PMC7176878.
Haploinsufficiency of NFKBIA reshapes the epigenome antipodal to the IDH mutation and imparts disease fate in diffuse gliomas. Cell reports. Medicine 2023 — PMC10314122.
MiR-196b-5p activates NF-κB signaling in non-small cell lung cancer by directly targeting NFKBIA. Translational oncology 2023 — PMC10458993.
PubMed PMID:1829648 — UniProt-cited evidence.
PubMed PMID:10637284 — UniProt-cited evidence.
PubMed PMID:14702039 — UniProt-cited evidence.