NCSTN / Nicastrin · IHC design guide

Design Immunohistochemistry for NCSTN

Plan chromogenic NCSTN IHC in paraffin sections around the cytoplasmic staining reported in most tissues (HPA tissue IHC). Use lung macrophages or pancreatic exocrine cells as high-staining reference populations (HPA tissue IHC), and keep fixation consistent when comparing sections.

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

Immunohistochemistry protocol sheet for NCSTN (IHC for NCSTN): expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC), antibody A03144, validated IHC image, and IHC protocol steps — Printable **NCSTN** IHC protocol sheet — expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC), antibody A03144, controls and protocol steps. Open the full NCSTN IHC guide →

Navigate to: Protocols Staining pattern Controls Antibodies

NCSTN 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 across most tissues (HPA tissue IHC)
	Antigen retrieval	Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen)
	Positive control ⓘ	Lung+4 more · see all
	Negative control ⓘ	Adipose tissue+4 more · see all

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 shows low consistency with RNA expression (HPA tissue IHC)
	Regulation	Specific expression regulation not reported (UniProt)
	Isoform / epitope	2 isoforms; check epitope against extracellular and cytoplasmic regions (UniProt)

Section 1

Recommended NCSTN IHC & IF Protocols

The catalog antibody’s IHC-P protocol is paired with published NCSTN IHC methods from two articles (PMC7092570; PMC11668073).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Tissue sections; selected-image fixative not specified (standard IHC workflow)
Fixation	Image fixative and duration unreported (datasheet A03144); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen)
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-NCSTN, 5 μg/mL (datasheet A03144)
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	NCSTN-positive staining in macrophages of lung (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control.

💡Decision noteStart with citrate pH 6.0 at 95–98 °C for 20 min (page retrieval rule); the articles also report EDTA or citric acid retrieval (PMC7092570; PMC11668073).

Section 2

What Is the Expected NCSTN Staining Pattern?

In paraffin sections, expect predominantly cytoplasmic NCSTN staining across many tissues, with strong staining reported in lung macrophages, pancreatic exocrine glandular cells and cells in testicular seminiferous ducts (HPA: tissue IHC). NCSTN has a large extracellular region, one transmembrane segment and a short cytoplasmic tail, so membrane-associated staining may also be plausible (UniProt Q92542 topology). Interpret intensity cautiously: HPA rates its tissue IHC profile Approved but reports low consistency with RNA expression (HPA: tissue IHC reliability).

What am I looking at on my slide?
Cytoplasmic staining in lung macrophages or pancreatic exocrine glandular cells, with stronger signal than nearby unstained cells (HPA: High in these cell types).	This matches reported tissue IHC. Score the named cell population and its compartment, rather than treating every cell in the section as an equivalent positive control (HPA: tissue IHC; general IHC practice).
Signal confined to nuclei, with no convincing cytoplasmic or membrane-associated pattern.	An exclusively nuclear pattern conflicts with HPA's cytoplasmic tissue profile and UniProt's membrane and vesicle annotations; investigate staining artefact before assigning it to NCSTN (HPA: tissue IHC; UniProt Q92542 subcellular location).
Strong staining in adipocytes, cardiomyocytes or skeletal myocytes instead of the expected positive cell populations (HPA: Not detected in these cells).	Consider cross-reactivity or endogenous detection activity, especially if the signal also appears in a no-primary control. HPA's low antibody–RNA consistency makes an unexpected result a finding to verify, rather than proof of either cause (HPA: tissue IHC reliability; general IHC practice).
Color spread broadly across tissue, including spaces or cell types without a coherent cellular pattern.	Treat diffuse signal as background until a no-primary control and inspection of blocking, washes and chromogen development support specific staining (general IHC practice). It does not match HPA's cell-associated cytoplasmic profile (HPA: tissue IHC).
No staining in lung macrophages or pancreatic exocrine glandular cells (HPA: High in these cell types).	A blank known-positive population raises a sensitivity or workflow concern; confirm that the cells are present, then assess antibody dilution, retrieval and detection with appropriate controls (HPA: tissue IHC; general IHC practice).

💡Expected NCSTN appearanceCall a section positive when identifiable lung macrophages, pancreatic exocrine glandular cells or cells in testicular seminiferous ducts show clear cytoplasmic staining at the reported High level; isolated nuclear signal or diffuse color across unrelated structures is suspect (HPA: tissue IHC; UniProt Q92542 subcellular location).

How each factor affects the staining
Cell population and comparator	HPA reports High staining in three named populations, Medium staining in selected glandular, respiratory epithelial and neuronal cells, and no detection in specified others. Choose and score the actual cell type within each section; a tissue name alone does not define every cell's expected result (HPA: tissue IHC).
Topology and compartment	NCSTN spans the membrane at residues 670–690, with residues 34–669 extracellular and 691–709 cytoplasmic (UniProt Q92542 topology). This supports a membrane-associated protein, while HPA describes the observed tissue IHC pattern as cytoplasmic; topology alone does not predict an antibody's visible outline (HPA: tissue IHC).
Evidence and antibody validation	HPA marks tissue IHC reliability Approved despite low staining–RNA consistency. It lists IHC approval for HPA054846 and CAB021982, without an IHC Enhanced designation in the supplied record. Treat unusual tissue results as requiring controls and independent confirmation (HPA: tissue IHC reliability; HPA: antibody validation).
IF/ICC Q: Should its pattern match IHC?	A: HPA reports approved cytosolic ICC-IF localization and images in HaCaT, SiHa and U2OS; HPA070642 has ICC approval, while the supplied IHC-approved antibodies are separately listed. Use that evidence to interpret IF/ICC images, without assuming identical antibody performance or prescribing an IF protocol here (HPA: subcellular ICC-IF; HPA: antibody validation).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Known-positive lung macrophages are blank (HPA: High in macrophages).	The target cells may be absent from the sampled field, or the IHC workflow may lack sensitivity; the image alone cannot distinguish these possibilities (HPA: tissue IHC; general IHC practice).	Verify macrophages morphologically, compare a known-positive section, and check retrieval, antibody dilution and detection controls using the chosen antibody's validated IHC conditions (general IHC practice).
Pancreatic exocrine glandular cells stain weakly despite an expected High HPA level (HPA: tissue IHC).	Section or assay variation may reduce contrast; HPA's tissue profile is an observed reference, not a guaranteed intensity for every preparation (HPA: tissue IHC reliability; general IHC practice).	Compare matched positive-control sections and review retrieval, dilution and development consistently before changing the biological interpretation (general IHC practice).
Signal appears mainly in nuclei.	Exclusive nuclear staining does not fit HPA's cytoplasmic IHC profile or UniProt's membrane and vesicle localization (HPA: tissue IHC; UniProt Q92542 subcellular location).	Inspect a no-primary control, confirm morphology and evaluate whether an independently validated IHC antibody reproduces the cellular pattern (general IHC practice; HPA: antibody validation).
Adipocytes or cardiomyocytes show strong signal (HPA: Not detected in these cells).	Cross-reactivity or endogenous detection activity is possible; HPA's low staining–RNA consistency prevents a definitive assignment from this discrepancy alone (HPA: tissue IHC reliability; general IHC practice).	Run a no-primary control, inspect signal location within the named cells and seek confirmation with another IHC-approved antibody when the result matters (general IHC practice; HPA: antibody validation).
Brown signal coats multiple structures without distinct stained cells.	Diffuse background can result from incomplete blocking or washes, or excessive chromogen development (general IHC practice).	Compare the no-primary slide, review blocking and wash steps, and shorten development if control background rises; then reassess whether cell-associated cytoplasmic signal remains (general IHC practice; HPA: tissue IHC).
A tissue reported as ‘Not detected’ contains a few stained cells.	HPA's negative entries name particular cell populations; for example, the adipose entry specifies adipocytes, so another cell type in that section cannot be classified from that entry alone (HPA: tissue IHC).	Identify the stained cells first, score them separately, and compare their pattern with a reported positive population and assay controls before calling the finding specific (HPA: tissue IHC; general IHC practice).

Source: UniProt Q92542 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing NCSTN staining

A03144 · IHCImmunohistochemistry of Nicastrin in human brain tissue with Nicastrin antibody at 5 μg/mL.

A03144 · IFImmunofluorescence of Nicastrin in Human Brain cells with Nicastrin antibody at 20 μg/mL.

IF analysis of Nicastrin/NCSTN using anti-Nicastrin/NCSTN antibody (A03144-1). Nicastrin/NCSTN was detected in an immunocytochemical section of SiHa cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-Nicastrin/NCSTN Antibody (A03144-1) overnight at 4°C. DyLight®594 Conjugated Goat Anti-Rabbit IgG (BA1142) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

A03144-1 · IFIF analysis of Nicastrin/NCSTN using anti-Nicastrin/NCSTN antibody (A03144-1). Nicastrin/NCSTN was detected in an immunocytochemical section

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000162736-NCSTN/tissue/cerebral+cortex#imid_17227615

HPA · CerebellumHuman Protein Atlas IHC, Cerebellum. Source: https://www.proteinatlas.org/ENSG00000162736-NCSTN/tissue/cerebellum#imid_17227618

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000162736-NCSTN/tissue/colon#imid_17227541

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000162736-NCSTN/tissue/liver#imid_17227526

HPA · ICC-IF HaCaTHuman Protein Atlas immunofluorescence, HaCaT, antibody HPA070642; green = NCSTN, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for NCSTN IHC & IF

🧪Run lung first and score its macrophages, which must show staining as the positive population (HPA: High in lung macrophages). Run adipose tissue as the negative comparator and score adipocytes (HPA: Not detected in adipocytes); on the lung slide, neighboring cells without target staining should show only counterstain, providing an internal background reference (standard IHC practice).

Positive control tissue: Lung (Macrophages, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show NCSTN in HaCaT, SiHa, U2OS, with annotated localisation: Cytosol (approved) (HPA subcellular).

Technical controls: Include a no-primary (secondary-only) control, a host-species-matched isotype control for a monoclonal antibody or nonimmune IgG for a polyclonal antibody, and identically processed NCSTN-knockout material or cognate-peptide competition when the immunogen is known (standard IHC practice). For lung IHC, block endogenous peroxidase and distinguish macrophage pigment from chromogen; for IF, assess macrophage autofluorescence (standard IHC/IF practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence, and the selected A03144 brain IHC caption does not state a fixative (A03144 caption: fixative unreported). Retrieval dependence is unreported, so assess antigen retrieval empirically on paraffin sections; the supplied evidence does not establish that frozen sections or IF/ICC are easier (supplied target/application evidence). Lung macrophage pigment can complicate chromogenic scoring, so compare morphology and the no-primary control (standard IHC practice).

HPA tissue IHC evidence for NCSTN

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low 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
Lung	Macrophages	High	Protein (IHC)	HPA →
Pancreas	Exocrine glandular cells	High	Protein (IHC)	HPA →
Testis	Cells in seminiferous ducts	High	Protein (IHC)	HPA →
Adrenal gland	Glandular cells	Medium	Protein (IHC)	HPA →
Appendix	Glandular cells	Medium	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	Adipocytes	Not detected	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	Not detected	Protein (IHC)	HPA →
Heart muscle	Cardiomyocytes	Not detected	Protein (IHC)	HPA →
Lymph node	Germinal center cells	Not detected	Protein (IHC)	HPA →
Oral mucosa	Squamous epithelial cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced NCSTN IHC Tips

Troubleshoot NCSTN staining in paraffin section chromogenic IHC, with one entry for IF/ICC experimental design.

How should I retrieve NCSTN antigen when paraffin sections stain weakly?

Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval setting). Keep section thickness, cooling time, antibody concentration, and detection conditions constant while comparing retrieved sections with a no retrieval control (standard IHC practice). Assess staining in lung macrophages or pancreatic exocrine glandular cells, both reported as high, alongside a negative reagent control (HPA: High in macrophages; High in exocrine glandular cells). If staining remains weak, test a different retrieval buffer in a separate titration, recording tissue integrity and background because the antibody’s epitope and retrieval tolerance are unspecified (record: epitope not supplied; standard IHC practice).

Could fixation explain weak or uneven NCSTN staining?

NCSTN-specific sensitivity to fixation is unknown from the supplied evidence, so treat any fixation effect as an experimental question (record: no target-specific fixation evidence supplied). The selected brain tissue IHC caption reports antibody use at 5 µg/mL but does not state the fixative (caption: A03144). Compare sections with documented fixation histories using the same citrate pH 6.0 retrieval, primary antibody concentration, and chromogenic detection settings (page retrieval setting; standard IHC practice). Include a no-primary control and inspect morphology before attributing uneven signal to fixation; differences in processing and staining conditions can also affect IHC appearance (standard IHC practice).

Where should convincing NCSTN staining appear in a tissue section?

Assess NCSTN primarily within the cytoplasmic or membrane-associated region, allowing a vesicular pattern rather than demanding an exclusively sharp cell-surface outline (UniProt Q92542: membrane and cytoplasmic vesicle membrane; HPA: cytoplasmic expression in most tissues). Its single transmembrane segment spans residues 670–690, with residues 34–669 extracellular and 691–709 cytoplasmic (UniProt Q92542 topology). HPA also reports an approved cytosolic subcellular location, so morphology and cell identity should guide interpretation of diffuse staining (HPA: Cytosol approved; standard IHC practice). Compare candidate positive cells with a no-primary control and examine whether staining follows intact cellular structures across the section (standard IHC practice).

How can an unknown epitope affect NCSTN IHC interpretation?

Confirm the antibody’s immunogen or mapped epitope before assigning staining to a particular NCSTN isoform; the record lists 2 isoforms but supplies no antibody epitope (UniProt Q92542: isoforms 1 and 2; record: epitope not supplied). NCSTN has a large extracellular region at residues 34–669, a cytoplasmic tail at 691–709, and 16 annotated glycosylation sites (UniProt Q92542 topology and glycosylation). These features help frame epitope-access questions, but they do not establish which sequence the catalog antibody recognizes (record: epitope not supplied). Compare staining after the stated citrate pH 6.0 retrieval with appropriate controls before interpreting differences between samples as isoform changes (page retrieval setting; standard IHC practice).

How should I design an NCSTN IF multiplex experiment?

For the separate IF/ICC workflow, pair NCSTN with a marker that identifies the expected cell type, then confirm that both signals occupy the intended cells (standard IF practice). HPA reports high tissue staining in lung macrophages and pancreatic exocrine glandular cells, providing cell identities to consider when selecting a tissue marker (HPA: High in macrophages; High in exocrine glandular cells). Choose a fluorophore channel with low tissue autofluorescence and include single-color and no-primary controls to assess bleed-through and background (standard IF practice). If the antibody epitope is on the cytoplasmic tail, validate permeabilisation; if it is on an accessible extracellular domain, compare permeabilised and nonpermeabilised conditions because this antibody’s epitope is unspecified (UniProt Q92542 topology; record: epitope not supplied; standard IF practice).

What should I check when NCSTN DAB staining is widespread?

Begin with a no-primary control to identify signal from the detection system, and check whether endogenous peroxidase blocking reduces DAB staining (standard chromogenic IHC practice). Titrate the catalog antibody around the tested brain tissue caption concentration of 5 µg/mL, recording signal and background under identical detection conditions (caption: A03144; standard IHC practice). Review blocking, wash steps, and exposure to chromogen if staining extends beyond intact cellular boundaries or accumulates at tissue edges (standard IHC practice). Because HPA describes cytoplasmic expression in most tissues yet low consistency between staining and RNA expression, widespread cytoplasmic color alone cannot establish NCSTN specificity (HPA: tissue profile and reliability description).

How should I quantify NCSTN staining across tissue samples? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and scoring area before comparing sections, then record an H-score or the percentage of positive cells using fixed intensity thresholds (standard quantitative IHC practice). For heterogeneous tissue, report positive-cell density per mm² as well and normalize counts to the analyzed viable tissue area or the number of eligible cells (standard quantitative IHC practice). Score cytoplasmic or membrane-associated staining within the prespecified cells while excluding folds, edges, and necrotic regions (UniProt Q92542: membrane and cytoplasmic vesicle membrane; HPA: cytoplasmic tissue profile; standard IHC practice). Keep retrieval, antibody concentration, imaging, and threshold settings consistent, and report scores by cell type because HPA levels differ across cell populations (HPA: tissue IHC profile; standard quantitative IHC practice).

How can I distinguish true NCSTN signal from staining artefacts?

Favor reproducible cellular staining in an expected compartment, such as cytoplasmic or vesicle-associated regions, over isolated precipitate or staining confined to a section edge (UniProt Q92542: membrane and cytoplasmic vesicle membrane; HPA: cytoplasmic tissue profile; standard IHC practice). Check whether candidate cells match a documented pattern, such as high staining in lung macrophages or pancreatic exocrine glandular cells (HPA: High in macrophages; High in exocrine glandular cells). Treat staining in necrotic areas or no-primary controls as suspect, and assess residual endogenous peroxidase activity before interpreting DAB color (standard chromogenic IHC practice). HPA rates the tissue staining Approved but reports low consistency with RNA expression, so use independent controls before claiming a tissue-specific biological difference (HPA: reliability and reliability description).

Boster reagents

Best NCSTN / Nicastrin IHC Antibodies

The rendered NCSTN antibodies have IHC data from human brain tissue and IF data from SiHa cells (catalog image captions); both list Human, Mouse, and Rat reactivity (catalog reactivity).

Real IHC data Immunohistochemistry of Nicastrin in human brain tissue with Nicastrin antibody at 5 μg/mL.

Anti-Nicastrin NCSTN Antibody

Cat # A03144

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Real IF data

Anti-Nicastrin/NCSTN Antibody ®

Cat # A03144-1

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A03144 is listed for IHC-P and IF, with an IHC image of human brain tissue at 5 μg/mL (catalog applications; A03144 IHC image caption). A03144-1 is listed for ICC and IF, with an IF image of SiHa cells at 5 μg/mL (catalog applications; A03144-1 IF image caption).

Which to pick: Choose A03144 for paraffin-section IHC: its IHC-P application and human brain tissue image support that choice, while the fixative is unreported (catalog applications; A03144 IHC image caption). Choose A03144-1 for IF/ICC when the SiHa cell example is relevant (catalog applications; A03144-1 IF image caption). Both list Human, Mouse, and Rat reactivity for cross-species planning; clonality is unspecified for both rendered SKUs, and their pictured results are limited to the samples named above (catalog reactivity and clone fields; image captions).

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

References

UniProt Consortium. UniProt entry Q92542 (NICA_HUMAN, Nicastrin).
Human Protein Atlas. NCSTN tissue IHC expression (reliability: Approved).
Human Protein Atlas. NCSTN subcellular location (ICC-IF): Localized to the cytosol..
Human Protein Atlas. NCSTN antibody validation summary (3 antibodies).
NCSTN promotes hepatocellular carcinoma cell growth and metastasis via β-catenin activation in a Notch1/AKT dependent manner. Journal of experimental & clinical cancer research : CR 2020 — PMC7339515.
Effect of nicastrin on hepatocellular carcinoma proliferation and apoptosis through PI3K/AKT signalling pathway modulation. Cancer cell international 2020 — PMC7092570.
Expression of nicastrin, NICD1, and Hes1 in NCSTN knockout mice: implications for hidradenitis suppurativa, Alzheimer's, and liver cancer. European journal of medical research 2024 — PMC11668073.
PubMed PMID:10993067 — UniProt-cited evidence.
PubMed PMID:12975309 — UniProt-cited evidence.
PubMed PMID:14702039 — UniProt-cited evidence.